Agonists of Neurotrophin Receptors and Their Use as Medicaments

ABSTRACT

The invention relates to compounds of Formula I: 
     
       
         
         
             
             
         
       
     
     and pharmaceutically acceptable salts and prodrugs thereof, wherein R 1 , R 2 , and R 3  are defined as set forth in the specification. The compounds are agonists of neurotrophin (such as nerve growth factor) receptors.

This application claims the benefit of the earlier filing date of U.S.Provisional Application No. 61/378,823, filed Aug. 31, 2010, theentirety of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention applies to the area of therapeutics for neurological,psychiatric disorders, and ageing. In particular, it relates to theneuroprotective effect of small molecule agonists of neurotrophin (NerveGrowth Factor (NGF) or Brain-Derived Neurotrophic Factor (BDNF))receptors and the use of those agonists as medicaments.

2. Background Art

Ageing, neurological and psychiatric disorders cause death and damage tonerve cells. Frequent and relevant damage to the nervous system canresult from neuronal degeneration, ischemia, inflammation, immuneresponses, trauma, and cancer, among other things. As a consequence ofthese, nerve cells can die within minutes or hours or survive thisinitial damage in an impaired state that activates neurodegeneration,ending equally in cellular death.

Given the importance of the nervous system in enabling basic motorskills and sensing, there exists an interest in finding therapeuticweapons to protect the nervous system.

Neuroprotection is focused on the preservation, recovery, cure, orregeneration of the nervous system, its cells, structure, and function(Vajda et al., 2002). A goal of neuroprotection is to prevent orminimize the effects of an original damage to the nervous system, or toprevent or minimize the consequences of endogenous or exogenous noxiousprocesses causing damage to axons, neurons, synapses, and dendrites.

Treatment strategies in general are frequently based on the modulationof a single proposed injury factor. Although such treatments can beshown to be beneficial in highly constrained animal models, they areless likely to prove efficacious in the more complex human disorder thatinvolves more variable degrees of injury severity in a geneticallydiverse population (Faden and Stoica, 2007). Importantly, since thepresumed mechanisms of neuronal death are both complex and varied, suchas oxidative stress, mitochondrial dysfunction, protein aggregation,apoptosis, and inflammation (Youdim et al., 2005), single compoundshaving multipotential effects on multiple injury mechanisms aredesirable.

Several neuroprotective drugs are under investigation including thefollowing classes: anti-inflammatory agents, N-methyl D-aspartate (NMDA)antagonists, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) antagonists, dexanabinol, sodium channel blockers,thyrotropin-releasing hormone (TRH), growth factors, glucocorticoids,caffeinol, opioid antagonists, apoptosis inhibitors, free radicaltrappers/scavengers, erythropoietin, calcium channel blockers, magnesiumsulfate, and statins.

The ability of these pharmacological agents to limit secondarybiochemical damage and cell death has been disappointing (Faden andStoica, 2007).

Neurotrophins are growth factors that regulate the development andmaintenance of the peripheral and the central nervous systems (Lewin andBarde, 1996). Nerve growth factor (NGF) is the first discovered and bestcharacterized member of the neurotrophin family, which includes otherstructurally related proteins, such as brain-derived neurotrophic factor(BDNF). Nerve growth factor (NGF) is a homodimeric protein from theneurotrophin family that plays a crucial role in neuronal survival,differentiation and growth (Levi-Montalcini, 1987) and binds twodistinct cellular receptors: the tyrosine kinase receptor TrkA and thep75 receptor (Chao, 2003). NGF-TrkA binding activates the intrinsictyrosine kinase of the receptor, causing tyrosine phosphorylation ofTrkA and associated signalling partners and therefore activatingpromotion of cell survival or differentiation (Kaplan and Miller, 2000).The p75 receptor is a member of the tumor necrosis factor receptorsuperfamily. Depending on the cellular environment and the type ofligand, p75 can act as transducer of pro-survival, pro-apoptotic, orpro-differentiation signals (Barker, 1998; Rabizadeh et al., 1999;Zaccaro et al., 2001; Saragovi and Zaccaro, 2002). Accordingly,depending on the metabolic route, binding to either TrkA or p75receptors may trigger signals, depending on the cell type considered,linked to, indistinctly, differentiation and/or cell survival.Neurotrophins act through two main signaling pathways: thephosphatidylinositol 3-kinase (PI3K)-AKT pathway and themitogen-activated protein kinase (MAPK)-MEK pathway (both pathways areinvolved in the inhibition of apoptosis). Neurotrophic factors are knownto act also on mature neurons and in particular on injured anddegenerative cells (Lindvall et al. 1994; Tuszynski and Gage 1995;Lykissas et al. 2007; Song et al. 2009).

The potential of NGF as a therapeutic agent for several diseases hasbeen indicated by several investigators. Such diseases includeneurodegenerative disorders, nerve inflammation and certain types ofcancers, multiple sclerosis, neuromyelitis optica, amyotrophic lateralsclerosis (ALS), Parkinson's disease, Alzheimer's disease, Friedreich'sataxia, Huntington's disease, Dementia with Lewy bodies, spinal muscularatrophy, major depressive disorder, schizophrenia, glaucoma orperipheral neuropathies (diabetic or AIDS neuropathy) (Longo et al,2007; Schulte-Herbruggen, 2007; Shi, 2007; Hellveg, 2008; Shoval, 2005;Apfel, 2002; Anand, 2004). NGF has significant immunoregulatoryproperties during CNS inflammation to contribute to the maintenance ofthe CNS privilege (Villoslada and Genain, 2004). During ExperimentalAutoimmune Encephalomyelitis (EAE) in marmoset, NGF was able to inhibitthe development of the clinical symptoms when administeredintracerebroventricularly by continuous infusion apparently because ofits ability to induce an immunosuppressive microenvironment in the CNSwhich leads to decreased CNS infiltration (Villoslada et al., 2000). TheEnding that NGF induces immunosuppression during autoimmunedemyelination in addition to its neuroprotective properties in neuronsand oligodendrocytes makes it a very good candidate for the treatment ofCNS inflammatory diseases like MS. However, NGF is not the ideal drugcandidate due to its inability to cross the blood-brain barrier (BBB)(Poduslo and Curran, 1996), its short half life and its side effects(Apfel, 2002). Much effort has been made in the search for smallmolecules with NGF agonist activity, with better pharmacokinetics andless side effects. To achieve this goal, different approaches have beenattempted (Poduslo and Curran, 1996; Longo et al., 1997; Maliartchouk etal., 2000a; Maliartchouk et al., 2000b; Peleshok and Saragovi, 2006).

As such, there is an ongoing need for providing drugs, particularly NGFmimetics, with neuroprotective properties, which have preferablymultipotential effects, but without the drawbacks of NGF. The presentinventors have developed a family of compounds distinct from thosedisclosed in the art. The family of compounds of the invention arepeptidomimetics of neurotrophins (NGF, BDNF), and agonists to TrkA,TrkB, and p75 specific receptors. Some of the compounds of the inventionpromote, as a way of example, cell survival to an extent even higherthan NGF itself. The compounds of the invention are consideredpeptide-mimetics of neurotrophin and they all share a structure ofN-alkylglycine trimers.

SUMMARY OF THE INVENTION

The present invention is related to the use of peptoid compounds ofFormulae I-V, below, and the pharmaceutically acceptable salts andprodrugs thereof, as agonists of neurotrophin receptors, andspecifically nerve growth factor (NGF) receptors and brain-derivedneurotrophic factor (BDNF) receptors.

Compounds useful in the present invention have not been heretoforereported. Thus, one aspect of the present invention is directed to novelcompounds of Formulae I-V, as well as their pharmaceutically acceptablesalts and prodrugs.

Another aspect of the invention is directed to the use of compounds ofany of Formulae I-V, and their pharmaceutically acceptable salts andprodrugs, as agonists of NGF receptors. Another aspect of the inventionis directed to the use of compounds of any of Formulae I-V, and theirpharmaceutically acceptable salts and prodrugs, as agonists of BDNFreceptors.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use as a medicament. In one aspect of the invention, the medicamentis for use in preventing or treating nerve cell death or damage. In oneaspect of the invention, the medicament is for use in neuroprotection.In one aspect of the invention, the medicament is for use inregeneration of nerve cells. In one aspect of the invention, themedicament is for use in neuroenhancing. In one aspect, the medicamentis for use in preventing or treating a neurological or a psychiatricdisease. In one aspect of the invention, the medicament is for use inpreventing or treating a disease selected from the group consisting of aneurological disease, a preferentially neurodegenerative disorder (suchas amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, Friedreich's ataxia, Huntington's disease, Dementia with Lewybodies, and spinal muscular atrophy), nerve inflammation (such asmultiple sclerosis and neuromyelitis optica), major depressive disorder,schizophrenia, glaucoma, peripheral neuropathy (such as diabetic or AIDSneuropathy), and cancer (such as glioblastoma, astrocytoma,meduloblastoma, neurinoma, neuroblastoma, meningioma, colon cancer,pancreatic cancer, breast cancer, prostate cancer, leukemia, acutelymphocytic leukemia, osteosarcoma, hepatocellular carcinoma, ovariancarcinoma, lung adenocarcinoma, and esophagic carcinoma).

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use as a medicament for treating multiple sclerosis.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use as a medicament, wherein the medicament is a neuroregenerativedrug.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use as a medicament, wherein the medicament is an immunomodulator.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use as a medicament, wherein the medicament has a combination ofneuroprotective and immunomodulatory effects.

A further aspect of the present invention is to provide a pharmaceuticalcomposition, comprising a therapeutically effective amount of at leastone compound of any of Formulae I-V, or a pharmaceutically acceptablesalt or prodrug thereof, and a pharmaceutically acceptable carrier.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof in the manufacture of a medicament for preventing or treatingnerve cell death or damage.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament for providingneuroprotection.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament for the regeneration ofnerve cells.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament for preventing or treating aneurological disease or a psychiatric disease.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament for preventing or treating adisease selected from the group consisting of a neurological disease, apreferentially neurodegenerative disorder (such as amyotrophic lateralsclerosis (ALS), Parkinson's disease, Alzheimer's disease, Friedreich'sataxia, Huntington's disease, Dementia with Lewy bodies, and spinalmuscular atrophy), nerve inflammation (such as multiple sclerosis andneuromyelitis optica), major depressive disorder, schizophrenia,glaucoma, peripheral neuropathy (such as diabetic or AIDS neuropathy),and cancer (such as glioblastoma, astrocytoma, meduloblastoma,neurinoma, neuroblastoma, meningioma, colon cancer, pancreatic cancer,breast cancer, prostate cancer, leukemia, acute lymphocytic leukemia,osteosarcoma, hepatocellular carcinoma, ovarian carcinoma, lungadenocarcinoma, and esophagic carcinoma).

A further aspect of the invention is to provide the use of a compound ofany of Formulae or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament for preventing or treatingmultiple sclerosis.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a neuroregenerative drug.

A further aspect of the invention is to provide the use of a compound ofany of Formulae or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of an immunomodulator.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a medicament having a combination ofneuroprotective and immunomodulatory effects.

A further aspect of the invention is to provide the use of a compound ofany of Formulae I-V, or a pharmaceutically acceptable salt or prodrugthereof, in the manufacture of a neuroenhancing drug.

A further aspect of the invention is to provide a method for preventingor treating nerve cell death or damage, comprising administering to asubject in need thereof an effective amount of a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,or an effective amount of a pharmaceutical composition comprising acompound of any of Formulae I-V, or a pharmaceutically acceptable saltor prodrug thereof.

A further aspect of the invention is to provide a method for providingneuroprotection, comprising administering to a subject in need thereofan effective amount of a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof, or an effectiveamount of a pharmaceutical composition comprising a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a method for providingimmunomodulation, comprising administering to a subject in need thereofan effective amount of a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof, or an effectiveamount of a pharmaceutical composition comprising a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a method forregenerating nerve cells, comprising administering to a subject in needthereof an effective amount of a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof, or an effectiveamount of a pharmaceutical composition comprising a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a method for preventingor treating a disease selected from the group consisting of aneurological disease, a preferentially neurodegenerative disorder (suchas amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, Friedreich's ataxia, Huntington's disease, Dementia with Lewybodies, and spinal muscular atrophy), nerve inflammation (such asmultiple sclerosis and neuromyelitis optica), major depressive disorder,schizophrenia, glaucoma, peripheral neuropathy (such as diabetic or AIDSneuropathy), and cancer (such as glioblastoma, astrocytoma,meduloblastoma, neurinoma, neuroblastoma, meningioma, colon cancer,pancreatic cancer, breast cancer, prostate cancer, leukemia, acutelymphocytic leukemia, osteosarcoma, hepatocellular carcinoma, ovariancarcinoma, lung adenocarcinoma, and esophagic carcinoma), comprisingadministering to a subject in need thereof an effective amount of acompound of any of Formulae I-V, or a pharmaceutically acceptable saltor prodrug thereof, or an effective amount of a pharmaceuticalcomposition comprising a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a method for preventingor treating multiple sclerosis, Alzheimer's disease, Parkinson'sdisease, or glaucoma, comprising administering to a subject in needthereof an effective amount of a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof, or an effectiveamount of a pharmaceutical composition comprising a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a method of treating adisease responsive to the stimulation of the activity of neurotrophin,or a neurotrophin receptor, in a mammal suffering from lack ofstimulation thereof, comprising administering an effective amount acompound of any of Formulae I-V, or a pharmaceutically acceptable saltor prodrug thereof.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use in stimulating the activity of neurotrophin, or a neurotrophinreceptor.

A further aspect of the present invention is to provide a method ofstimulating neurotrophin receptor activity in a subject in need thereof,comprising administering a compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof, to the subject. Inone embodiment, the neurotrophin receptor is TrkA receptor, TrkBreceptor, or p75 receptor.

A further aspect of the invention is to provide a method of treating adisease responsive to the stimulation of the activity of nerve growthfactor, or a nerve growth factor receptor, in a mammal suffering fromlack of stimulation thereof, comprising administering an effectiveamount a compound of any of Formulae I-V, or a pharmaceuticallyacceptable salt or prodrug thereof.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use in stimulating the activity of nerve growth factor, or a nervegrowth factor receptor.

A further aspect of the present invention is to provide a method ofstimulating nerve growth factor receptor activity in a subject in needthereof, comprising administering a compound of any of Formulae I-V, ora pharmaceutically acceptable salt or prodrug thereof, to the subject.In one embodiment, the nerve growth factor receptor is TrkA receptor orp75 receptor.

A further aspect of the invention is to provide a method of treating adisease responsive to the stimulation of the activity of brain-derivedneurotrophic factor, or a brain-derived neurotrophic factor receptor, ina mammal suffering from lack of stimulation thereof, comprisingadministering an effective amount a compound of any of Formulae I-V, ora pharmaceutically acceptable salt or prodrug thereof.

A further aspect of the invention is to provide a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,for use in stimulating the activity of brain-derived neurotrophicfactor, or a brain-derived neurotrophic factor receptor.

A further aspect of the present invention is to provide a method ofstimulating brain-derived neurotrophic factor receptor activity in asubject in need thereof, comprising administering a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof,to the subject. In one embodiment, the nerve growth factor receptor isTrkB receptor or p75 receptor.

A further aspect of the present invention is to provide a method ofpreparing a pharmaceutical composition, comprising admixing an effectiveamount of a compound of any of Formulae I-V, or a pharmaceuticallyacceptable salt or prodrug thereof, with a pharmaceutically acceptablecarrier.

Additional embodiments and advantages of the invention will be set forthin part in the description that follows, and will flow from thedescription, or may be learned by practice of the invention. Theembodiments and advantages of the invention will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims.

It is to be understood that both the foregoing summary and the followingdetailed description are exemplary and explanatory only and notrestrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 depicts the results of Example 2 on G79, G80, and G81 in the doseresponse differentiation assay in PC12 cell line. FIG. 1A. depictsimages of PC12 cells differentiated in the presence of G79. FIG. 1Bdepicts images of PC12 cells differentiated in the presence of G80. FIG.1C depicts images of PC12 cells differentiated in the presence of G81.FIG. 1D shows the number of differentiated cells in the dose responsedifferentiation assay. FIG. 1E shows the percentage of differentiatedcells calculated relative to NGF induced differentiation. Data are themean±SEM of at least three experiments, each in duplicate.

FIG. 2 shows effects of G79, G80, and G81 in promotion of RN22 cellsurvival. Depicted are the means±S.E. of three experiments, each induplicate. *p<0.05, **p<0.01 (ANOVA) respect to stress control.

FIG. 3 depicts the effects of G79, G80, and G81 in secretagogue activityassay (FIG. 3A), in synergistic activity assay (FIG. 3B), in TrkAactivation assay (FIG. 3C), in signalling inhibition assay in thepresence of LY294002 (FIG. 3D), and in signalling inhibition assay inthe presence of PD98059 (FIG. 3E). FIG. 3F-3J show the effects depictedin FIG. 3A-3E, respectively, as percentages of differentiated cellscalculated relative to NGF induced differentiation.

FIG. 4 depicts the results of G79 in a glaucoma model. FIG. 4A shows thecount of the retinal ganglion cells for control, placebo, 200 μg/ml NGF,200 μg/ml G79, and 400 μg/ml G79. FIG. 4B shows retina sections aftertreatment with control, placebo, 200 μg/ml NGF, 200 μg/ml G79, and 400μg/ml G79. FIG. 4C shows the count of the retinal ganglion cells forcontrol, placebo, 200 μg/ml G79, Timolol, and 200 μg/ml G79 and Timolol.

FIG. 5 depicts the results of G79, G80, and G81 in Parkinson's diseasemodel (FIG. 5A) and in oxidative stress model (FIG. 5B).

FIG. 6 depicts the fluorophoric images of the results of the Westernblot analysis of PC12 cells treated with 100 ng/ml of G79.

FIG. 7 shows the intracellular pathway activated by the phosphorylationof the receptor tyrosine kinase (RTK).

FIG. 8 depicts the effects of G79 and BDNF on the levels of activationof the phosphoproteins ATF-2, HSP-27, JNK, and STAT-1 tested by Luminextechnology.

FIG. 9 depicts the effects of G79 in the cytometry binding assay ofExample 13. FIG. 9A shows the mean channel fluorescence (MCF) in thecompetition assay in PC12 (NGF/G79-FITC). FIG. 9B shows the MCF in thecompetition assay in PC12 (NGF/G79-FITC) with anti-TrkA antibody. FIG.9C shows the MCF in the competition assay in PC12 (NGF/G79-FITC) withanti-p76 antibody. FIG. 9D shows the MCF in the competition assay inSH-SY5Y (BDNF/G79-FITC).

FIG. 10 depicts the results of G79, G80, and G81 in in vitro model ofamyotrophic lateral sclerosis (ALS).

FIG. 11 shows the results of G79, Gambogic amide and Xaliproden in thepreventative application in in vivo model of MS after i.p.administration of G79 or Gambogic amide (FIG. 11A) and after oraladministration of Xaliproden (FIG. 11B).

FIG. 12 shows the results of G79, Gambogic amide, and Xaliproden in thecurative application in in vivo model of MS after oral administration.

FIG. 13 depicts the results of the effect of G79 in the induction of theenzyme iNOS over 48 hours.

FIG. 14 depicts the results of G79 in the in vitro model forneuroinflammation. FIG. 14A shows the production of TNFα in cerebellarorganitypic culture and FIG. 14B shows the production of IL-1β inorganotypic cerebellar culture.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention is based on the use of compounds of FormulaeI-V, and the pharmaceutically acceptable salts and prodrugs thereof, asagonists of neurotrophin receptors, and especially agonists of nervegrowth factor (NGF) receptors and brain-derived neurotrophic factor(BDNF) receptors. In view of this property, compounds of Formulae I-V,and the pharmaceutically acceptable salts and prodrugs thereof, areuseful for preventing or treating diseases responsive to the stimulationof neurotrophin receptors, and especially nerve growth factor or a nervegrowth factor receptor, or brain-derived neurotrophic factor or abrain-derived neurotrophic factor receptor.

Compounds of any of Formula I-V show a good NGF like activity “in vitro”by inducing differentiation of PC12 cells and promoting cell survival ofRN22 cells and, therefore, have neuroprotective properties.

The compounds useful in this aspect of the invention are compoundsrepresented by Formula I:

and pharmaceutically acceptable salts and prodrugs thereof, wherein

R₁ is phenyl substituted with halogen or trifluoromethyl, and furtheroptionally substituted with one or two substituents selected from thegroup consisting of halogen, C₁₋₆ alkyl, (C₁₋₆)alkoxy, andhalo(C₁₋₆)alkyl; or

R₁ is pyrrolidin-1-yl;

R₂ is 2-oxo-pyrrolidin-1-ylmethyl or sulfamoylphenyl; and

R₃ is chosen from propyl, 1-methylethyl, butyl, 2-methylpropyl, pentyl,1-methylbutyl, 2-methylbutyl, hexyl, 4-methylpentyl, 3-methylpentyl,2-methylpentyl, and 1-methylpentyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is fluorophenyl. In one embodiment,the fluorophenyl group is 2-fluorophenyl, 3-fluorophenyl or4-fluorophenyl. Preferably, R₁ is 2-fluorophenyl. In one embodiment, thefluorophenyl group is further substituted with one or two substituentsselected from the group consisting of halogen, C₁₋₆ alkyl, (C₁₋₆)alkoxy,and halo(C₁₋₆)alkyl; preferably one or two substituents selected fromthe group consisting of halogen, C₁₋₄ alkyl, (C₁₋₄)alkoxy, andhalo(C₁₋₄)alkyl; more preferably one or two substituents selected fromthe group consisting of halogen, methyl, ethyl, propyl, isopropyl,methoxy, ethoxy, fluoromethyl, and trifluoromethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is chlorophenyl. In one embodiment,the chlorophenyl group is 2-chlorophenyl, 3-chlorophenyl or4-chlorophenyl. In one embodiment, R₁ is 2-chlorophenyl. In oneembodiment, the chlorophenyl group is further substituted with one ortwo substituents selected from the group consisting of halogen, C₁₋₆alkyl, (C₁₋₆)alkoxy, and halo(C₁₋₆)alkyl; preferably one or twosubstituents selected from the group consisting of halogen, C₁₋₄ alkyl,(C₁₋₄)alkoxy, and halo(C₁₋₄)alkyl; more preferably one or twosubstituents selected from the group consisting of halogen, methyl,ethyl, propyl, isopropyl, methoxy, ethoxy, fluoromethyl, andtrifluoromethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is bromophenyl. In one embodiment,the bromophenyl group is 2-bromophenyl, 3-bromophenyl or 4-bromophenyl.In one embodiment, R₁ is 2-bromophenyl. In one embodiment, thebromophenyl group is further substituted with one or two substituentsselected from the group consisting of halogen, C₁₋₆ alkyl, (C₁₋₆)alkoxy,and halo(C₁₋₆)alkyl; preferably one or two substituents selected fromthe group consisting of halogen, C₁₋₄ alkyl, (C₁₋₄)alkoxy, andhalo(C₁₋₄)alkyl; more preferably one or two substituents selected fromthe group consisting of halogen, methyl, ethyl, propyl, isopropyl,methoxy, ethoxy, fluoromethyl, and trifluoromethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is iodophenyl. In one embodiment, theiodophenyl group is 2-iodophenyl, 3-iodophenyl or 4-iodophenyl. In oneembodiment, R₁ is 2-iodophenyl. In one embodiment, the iodophenyl groupis further substituted with one or two substituents selected from thegroup consisting of halogen, C₁₋₆ alkyl, (C₁₋₆)alkoxy, andhalo(C₁₋₆)alkyl; preferably one or two substituents selected from thegroup consisting of halogen, C₁₋₄ alkyl, (C₁₋₄)alkoxy, andhalo(C₁₋₄)alkyl; more preferably one or two substituents selected fromthe group consisting of halogen, methyl, ethyl, propyl, isopropyl,methoxy, ethoxy, fluoromethyl, and trifluoromethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is trifluoromethylphenyl. In oneembodiment, the trifluoromethylphenyl group is 2-trifluoromethylphenyl,3-trifluoromethylphenyl or 4-trifluoromethylphenyl. Useful compoundsinclude those where R₁ is 2-trifluoromethylphenyl. In one embodiment,the trifluoromethylphenyl group is further substituted with one or twosubstituents selected from the group consisting of halogen, C₁₋₆ alkyl,(C₁₋₆)alkoxy, and halo(C₁₋₆)alkyl; preferably one or two substituentsselected from the group consisting of halogen, C₁₋₄ alkyl, (C₁₋₄)alkoxy,and halo(C₁₋₄)alkyl; more preferably one or two substituents selectedfrom the group consisting of halogen, methyl, ethyl, propyl, isopropyl,methoxy, ethoxy, fluoromethyl, and trifluoromethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₁ is pyrrolidin-1-yl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₂ is 2-oxo-pyrrolidin-1yl-methyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, wherein R₂ is sulfamoylphenyl. In oneembodiment, the sulfamoylphenyl group is 2-sulfamoylphenyl,3-sulfamoylphenyl, or 4-sulfamoylphenyl. Preferably, R₂ is4-sulfamoylethyl.

In one embodiment, compounds useful in the present invention arecompounds of Formula I, where R₃ is 2-methylpropyl, having the FormulaII:

and pharmaceutically acceptable salts and prodrugs thereof, wherein R₁is and R₂ are as defined above for Formula I.

In one embodiment, compounds useful in the present invention arecompounds of Formulae I-II, and pharmaceutically acceptable salts andprodrugs thereof, wherein R₁ is 2-fluorophenyl or pyrrolidin-1-yl, andR² is 2-oxo-pyrrolidin-1-ylmethyl or 4-sulfamoylphenyl as follows:

-   -   R₁:

-   -   R₂:

Preferred compounds according to present invention are compounds ofFormula I, represented by any of the following Formulae III-V, and theirpharmaceutically acceptable salts and prodrugs:

Formula III:

[N-(2-(2′-Fluorophenyl)ethyl)glycyl-[N-(2-methylpropyl)glycyl]-N-[3-(2′-oxopyrrolidinyl)propyl]glycinamide(G79);

Formula IV:

[N-(2-(2′-Fluorophenyl)ethyl)glycyl]-[N-(2-methylpropyl)glycyl]-N-[2-(4′-sulfamoylphenyl)ethyl]glycinamide(G80); and

Formula V:

[N-(2-(1-Pyrrolidinyl)ethyl)glycyl]-[N-(2-methylpropyl)glycyl]-N-[2-(4′-sulfamoylphenyl)ethyl]glycinamide(G81),and pharmaceutically acceptable salts and prodrugs thereof,

In one embodiment, the compound of Formula I is the compound of FormulaIII, or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the compound of Formula I is the compound of FormulaIV, or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the compound of Formula I is the compound of FormulaV, or a pharmaceutically acceptable salt or prodrug thereof.

Useful halogen groups include fluorine, chlorine, bromine and iodine.

Useful alkyl groups are selected from straight-chained and branched C₁₋₆alkyl groups, and more preferably straight chain C₁₋₄ alkyl groups andbranched chain C₁₋₄ alkyl groups. Typical C₁₋₆ alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,iso-butyl, 3-pentyl, hexyl, among others.

Useful halo(C₁₋₆)alkyl groups include any of the above-mentioned C₁₋₆alkyl groups substituted by one or more fluorine, chlorine, bromine oriodine atoms (e.g., fluoromethyl, difluoromethyl, trifluoromethyl,pentafluoroethyl, 1,1-difluoroethyl and trichloromethyl groups).Preferably, the halo(C₁₋₆)alkyl group is trifluoromethyl.

Useful C₁₋₆ alkoxy groups include oxygen substituted by one of the C₁₋₆alkyl groups mentioned above (e.g., methoxy, ethoxy, propoxy,iso-propoxy, butoxy, tert-butoxy, iso-butoxy, sec-butoxy, andpentyloxy).

It has been found that the compounds of Formula III-V inducedifferentiation in PC12 cells and survival of RN22 cells after stressinduction. The mechanism of action of the compounds of Formulae III-Vhas been investigated. Accordingly, the effect of the compounds ofFormulae III-V together with NGF in culture was tested. If the compoundswork as ligands or interfere in the pathway induced by NGF, they mayinhibit the activity of NGF. Otherwise if the compounds produce anadditive effect, they may work through a parallel mechanisms. As aresult, we found that G79, G80 and G81 do not produce an additive effectin combination with NGF. There is not a big difference in the number ofdifferentiated cells in the treatment with only the compound compared tothe combination treatment. It means that the compounds of Formulae III-Vact in the same pathway of NGF. The reduction in the NGF neurotrophicresponse correspond to a profile of a partial agonist of NGF.

To better understand the binding of the compounds of Formulae III-V tothe cell surface, PC12 cells were treated with NGF or the compounds inthe presence of the antibody anti-TrkA that blocks the binding site ofTrkA for NGF in the extracellular domain. Therefore, if peptidomimeticsexert their activity by activating the binding site, the presence ofthis antibody will prevent their activity. Otherwise, if the activity onNGF pathway is maintained, it will indicate that neurotrophinpeptidomimetics exert their activity by activating TrkA in other pointof the molecule different of the binding site of TrkA. The results showsthat while NGF neurotrophic activity was reduced by adding into theculture the antibody anti-TrkA, the percentage of differentiationbetween the treatment with the compounds alone or the compounds incombination with the antibody was not very different. It may suggestthat the activity of the compounds is not mediated by the binding to theextracellular portion of the TrkA receptor. However, this result doesnot exclude that the compounds of Formulae III-V bind either theintracellular portion of the TrkA or other members of intracellularsignalling pathway. It was also found that G79 competes with NGF andBDNF for the binding to neurotrophin receptors. This effect may be dueto the competition on the p75 receptor, which is much higher expressedon the cell surface of the cell lines that were used in the experiments.

The possibility that the compounds of Formulae III-V may act assecretagogues, inducing their effects through the up-regulation of thesynthesis of NGF was also considered. It was found that while thetreatment of NGF together with the antibody anti-NGF induces a reductionin the differentiation of PC12 cells, there is no difference in thepercentage of differentiation induced by G79, G80 and G81 in thepresence of the same antibody compared to the treatment with thecompounds alone. For this reason, G79, G80, and G81 do not act assecretagogues.

To study the mechanism of action of the compounds of Formulae III-V, itwas investigated if the neurotrophic activity of the molecules dependson AKT and ERK activation, the two main pathways of TrkA signaling. Itwas found that as the treatment with NGF in combination with theinhibitors LY294002 or PD98059 reduces the percentage of differentiationin PC12 cells compared to the treatment with only NGF, also thepercentage of differentiation with G79, G80 and G81 in combination withthe inhibitors is reduced compared to the treatment with the compoundsalone.

As a conclusion, the NGF-like small molecules of the present inventioninduce differentiation without activating the synthesis of NGF. Tneyalso act through the activation of PI3K/AKT and MAPK/ERK TrkA pathwayswithout binding to the extracellular portion of TrkA.

The ability of the compounds of Formula III-V to activate theneurotrophin pathways was tested in differentiation assays in thepresence of inhibitors of Pi3K and MAPkinase pathways together withLuminex assays. According to the results, compounds G79, G80, and G81activate the neurotrophin pathway. For example, heat-shock proteins suchas HSP-27, can be activated by different agents and neurotrophic factors(Liu H. et al, J. Neurochem., 86, 1553-1563, 2003; Yuan Y et al, Eur. J.Pharmacol. 586, 100-105, 2008; O'Reilly A M et al, Mol Neurobiol, 42,124-132, 2010). The conducted tests show that HSP-27 is significantlyactivated after G79 in vitro stimulation, similarly to NGF.

The effect of the compounds of Formula III-V on in vivo and in vitromodels of neurodegenerative disease was also tested. The neuroprotectiveeffect of G79 in the in vitro models of PD, ALS and neuroinflammationwas shown. Also, G79 showed beneficial effect in vivo in the animalmodels of both MS and glaucoma ameliorating the clinical score of theanimals affected by EAE, and reducing cell death of RGC in glaucomatouseyes. The penetration through blood-brain-barrier (BBB) of the compoundsof Formula III-V was also tested. According to the results, G79 showsbetter penetration through the BBB by active transport, explaining itsbetter profile than G80 and G81. This form of transport guarantees amore specific delivery of the drug and permanence inside the brain,compared to the passive transport.

In conclusion, the results show that neurotrophin-like molecules canprovide a good therapeutic strategy to overcome the problem of deliveryof neurotrophins into the brain, preserving, and even enhancing, thebeneficial effects of neurotrophins themselves for the treatment ofneurodegenerative diseases.

The term “prodrug”, as used herein, includes any compound derived fromthe compounds of any of Formulae I-V, for example, the ester, amide,phosphate, etc., which, upon being administered to an individual, iscapable of providing the compounds of any of Formulae I-V or thepharmaceutically acceptable salt thereof, directly or indirectly, tosaid individual. Preferably, said derivative is a compound thatincreases the bioavailability of the compounds of any of Formulae I-Vwhen administered to an individual or that promotes the release of thecompounds of any of Formulae I-V in a biological compartment. The natureof said derivative is not critical, provided that it may be administeredto an individual and that it provides the compounds of any of FormulaeI-V in an individual's biological compartment. The preparation of saidprodrug may be performed by conventional methods known by those skilledin the art. Conventional procedures for the selection and preparation ofsuitable prodrug derivatives are described in, for example, Design ofProdrugs, H. Bundgaard ed., Elsevier (1985). An example of prodrug ofthe compounds of any of Formulae I-V can be their encapsulation intoliposomes. By this procedure, the peptoid is treated with theappropriate liposome precursor (combination of a phospholipid likelecithin, cholesterol and water) in order to be encapsulated. Dependingupon the lipophilicity of the peptoid, the compound will be retained atthe lipophilic part of the liposome or at the aqueous inner portion.Regarding the therapeutic polymer, the peptoid could be attached to thepolymer by covalent bonds created after regioselective hydrolysis of theterminal carboxamide. This hydrolysis renders a free carboxylic acidthat can be condensed with an amino or hydroxyl activated group of thepolymer.

The term “pharmaceutically acceptable” means that a compound orcombination of compounds is sufficiently compatible with the otheringredients of a formulation, and not deleterious to the patient up tothose levels acceptable by the industry standards.

For therapeutic use, salts of the compounds of any of Formulae I-V arethose wherein the counter-ion is pharmaceutically acceptable.

The term “salt” as mentioned herein is meant to comprise any stablesalts, which the compounds of any of Formulae I-V are able to form.Preferred are the pharmaceutically acceptable salts. Salts that are notpharmaceutically acceptable are also embraced in the scope of thepresent invention, since they refer to intermediates that may be usefulin the preparation of compounds with pharmacological activity.

The salts can conveniently be obtained by treating the base form of thecompounds of any of Formulae I-V with such appropriate acids asinorganic acids such as hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

The pharmaceutically acceptable salts can be obtained by treating thebase form of the compounds of any of Formulae I-V with such appropriatepharmaceutically acceptable acids like inorganic acids, for example,including hydrochloric, hydrobromic and the like; sulfuric acid; nitricacid; phosphoric acid and the like; or organic acids, for example,acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxopropanoic,oxalic, malonic, succinic, maleic, fumaric, malic, tartaric,2-hydroxy-1,2,3-propane-tricarboxylic, methanesulfonic, ethanesulfonic,benzenesulfonic, 4-methylbenzene-sulfonic, cyclohexanesulfamic,2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and the like acids.

Conversely the salt form can be converted by treatment with alkali intothe free base form.

The term “pharmaceutical composition” means for the purpose of thepresent invention any composition which comprises as an active compound,to which is attributed, fully or in part, the therapeutic (e.g.pharmaceutical) effect, at least one of the compounds of the inventionor combinations thereof and that may optionally further comprise atleast one pharmaceutically acceptable non-active ingredient, as anexcipient, carrier or so.

The term “preventing” refers to keep from happening, existing, oralternatively delaying the onset or recurrence of a disease, disorder,or condition to which such term applies, or of one or more symptomsassociated with a disease, disorder, or condition. The term “prevention”refers to the act of preventing, as “preventing” is defined immediatelyabove.

The term “treating”, as used herein, refers to reversing, alleviating,or inhibiting the progress of the disorder or condition to which suchterm applies, or one or more symptoms of such disorders or condition.The term “treatment” refers to the act of treating, as “treating” isdefined immediately above.

The term “subject” means animals, in particular mammals such as dogs,cats, cows, horses, sheep, geese, and humans. Particularly preferredsubjects are mammals, including humans of both sexes.

An “effective amount” of the compounds of any of Formulae I-V andpharmaceutically acceptable salts or prodrugs thereof, may be in therange from 0.01 mg to 50 g per day, from 0.02 mg to 40 g per day, from0.05 mg to 30 g per day, from 0.1 mg to 20 g per day, from 0.2 mg to 10g per day, from 0.5 mg to 5 g per day, from 1 mg to 3 g per day, from 2mg to 2 g per day, from 5 mg to 1.5 g per day, from 10 mg to 1 g perday, from 10 mg to 500 mg per day.

Nerve cells include those cells from any region of the brain, spinalcord, optic nerve, retina, and peripheral ganglia. Neurons include thosein embryonic, fetal, or adult neural tissue, including tissue from thehippocampus, cerebellum, spinal cord, cortex (e.g., motor orsomatosensory cortex), striatum, basal forebrain (cholinergic neurons),ventral mesencephalon (cells of the substantia nigra), and the locusceruleus (neuroadrenaline cells of the central nervous system).

The invention also covers the use of the compounds of any of FormulaeI-V, and pharmaceutically acceptable salts and prodrugs thereof, asactive ingredients in the manufacture of medicaments for the preventionor treatment of nerve cell death or damage. In other words, the presentinvention relates to the compounds of any of Formulae I-V, andpharmaceutically acceptable salts and prodrugs thereof, for use in theprevention or treatment of nerve cell death or damage. Similarly, thepresent invention relates to a method of neuroprotection comprisingadministering to a subject in need thereof an effective amount of acompound of any of Formulae I-V, or a pharmaceutically acceptable saltor a prodrug thereof.

In one embodiment of the present invention, compounds of any of FormulaeI-V, and pharmaceutically acceptable salts and prodrugs thereof, may beused for the prevention or treatment of one or more, preferably two ormore, pathological or harmful conditions related to nerve cell death ordamage selected from, but not being limited to, chemical substances suchas oxidative stress conditions, toxic substances, infectious organisms,radiation, traumatic injury, hypoxia, ischemia, abnormal misfoldedproteins, excitotoxins, free radicals, endoplasmic reticulum stressors,mitochondrial stressors including but not limited to inhibitors of theelectron transport chain, Golgi apparatus antagonists, axonal damage orloss, demyelination, inflammation, pathological neuronal burst(seizures). Also preferably, the uses and methods of the presentinvention are directed to preventing or treating nerve cell death ordamage, regardless of cause.

The terms “neuroprotection”, “neuroprotective”, or “neuroprotectiveeffect” refer to the ability to prevent or reduce death or damage tonerve cells, including neurons and glia, or rescuing, resuscitating orreviving nerve cells, e.g., following in pathological or harmfulconditions to the brain, central nervous system or peripheral nervoussystem. Thus, this neuroprotective effect comprises the conferredability of neuronal cells to maintain or recover their neuronalfunctions. It stabilizes the cell membrane of a neuronal cell or helpsin the normalization of neuronal cell functions. It prevents the loss ofviability or functions of neuronal cells. It comprises the inhibition ofprogressive deterioration of neurons that leads to cell death. It refersto any detectable protection of neurons from stress. Neuroprotectionincludes the regeneration of nerve cells, i.e. the re-growth of apopulation of nerve cells after disease or trauma.

Currently the majority of the neurological and psychiatric diseaseslacks specific treatments aimed to stop or ameliorate the course of thedisease, which are called “disease modifying drugs”. This contrasts withthe symptomatic therapies which are common for such diseases but do notchange the course of the disease. A neuroprotective drug is a DiseaseModifying Drug (DMD) for the treatment of brain diseases.

As such, in one embodiment, the present invention relates to the use ofthe compounds of any of Formulae I-V, and pharmaceutically acceptablesalts and prodrugs thereof, as active ingredients in the manufacture ofa medicament for the regeneration of nerve cells. In other words, thepresent invention relates to the compounds of any of Formulae I-V, andpharmaceutically acceptable salts and prodrugs thereof, for use for theregeneration of nerve cells. Similarly, the present invention relates toa method of regenerating nerve cells, comprising administering to asubject in need thereof an effective amount of a compound of any ofFormulae I-V, or a pharmaceutically acceptable salt or prodrug thereof.

Neuroprotection may be determined directly by, for example, measuringthe delay or prevention of neuronal death, such as, for example, by areduction in the number of apoptotic neurons in cerebrocortical culturesfollowing a stress. Neuroprotection may also be determined directly by,for example, measuring the severity or extent of damage to, orfunctional loss by, a tissue or organ of the nervous system followingsuch a stress, such as, for example, by measuring a decrease in the sizeof brain infarcts after occlusion of the middle cerebral artery (MCAO)or reperfusion injury. Also, neuroprotection can be identified bymagnetic resonance imaging (measuring brain volume, tractography, levelsof N-acetyl-aspartate by spectroscopy, optic coherent tomography).Alternatively, neuroprotection may be determined indirectly by detectingthe activation of one or more biological mechanisms for protectingneurons, including, but not limited to, detecting activation of theKeap1/Nrf2 pathway or induction of one or more phase 2 enzymes,including but not limited to hemeoxygenase-1 (HO-1). Methods ofdetecting and measuring neuronal protection are provided in the Examplesbelow, and other such methods are known in the art.

The various uses and methods employing the compounds of any of FormulaeI-V, and pharmaceutically salts and/or prodrugs thereof, in the presentinvention comprise acute administration, i.e. occurring within severalminutes to about several hours from injury, or chronic administration,suitable for chronic neurological or psychiatric diseases.

In one embodiment of the present invention, in the various uses andmethods of neuroprotection or of prevention or treatment of nerve celldeath or damage, the compounds of any of Formulae I-V, andpharmaceutically acceptable salts and prodrugs thereof, are administeredto a subject with a neurological or psychiatric disease.

Neurological diseases are those disorders of the central and peripheralnervous system, including disorders of the brain, spinal cord, cranialnerves, peripheral nerves, nerve roots, autonomic nervous system,neuromuscular junction, and muscle.

Diseases of the central and peripheral nervous system, which may besubject of prevention and/or treatment according to present inventioninclude, without being limited to, as knowledge in clinicalmanifestations advances, Absence of the Septum Pellucidum, Acid LipaseDisease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, AcuteDisseminated Encephalomyelitis, Adie's Pupil, Adie's Syndrome,Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, AicardiSyndrome, Aicardi-Goutieres Syndrome Disorder, AIDS—NeurologicalComplications, Alexander Disease, Alpers' Disease, AlternatingHemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis,Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia,Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts,Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation,Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellaror Spinocerebellar Degeneration, Atrial Fibrillation and Stroke,Attention Deficit-Hyperactivity Disorder (ADHD), Autism, AutonomicDysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker'sMyotonia, Behcet's Disease, Bell's Palsy, Benign EssentialBlepharospasm, Benign Focal Amyotrophy, Benign IntracranialHypertension, Bernhardt-Roth Syndrome, Binswanger's Disease,Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus BirthInjuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brainand Spinal Tumors, Brain Aneurysm, Brain infarction, Brain ischemia,Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy,CADASIL, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas,Cavernous Angioma, Cavernous Malformation, Central Cervical CordSyndrome, Central Cord Syndrome, Central Pain Syndrome, Central PontineMyelinolysis, Cephalic Disorders, Ceramidase Deficiency, CerebellarDegeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, CerebralArteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, CerebralCavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, CerebralPalsy, Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-ToothDisease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea,Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy(CIDP), Chronic Orthostatic Intolerance, Chronic Fain, Cockayne SyndromeType II, Coffin Lowry Syndrome, COFS, Colpocephaly, Coma, ComplexRegional Pain Syndrome, Congenital Facial Diplegia, CongenitalMyasthenia, Congenital Myopathy, Congenital Vascular CavernousMalformations, Corticobasal Degeneration, Cranial Arteritis,Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative TraumaDisorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease,Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome,Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Deep BrainStimulation for Parkinson's Disease, Dejerine-Klumpke Palsy, Dementia,Dementia—Multi-Infarct, Dementia—Semantic, Dementia—Subcortical,Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, DentatorubralAtrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome,Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Dysautonomia,Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia CerebellarisMyoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, EarlyInfantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis,Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy,familial infantile, with intracranial calcification and chroniccerebrospinal fluid lymphocytosis; Cree encephalitis; Pseudo-Torchsyndrome; Pseudotoxoplasmosis syndrome, EncephalotrigeminalAngiomatosis, Epilepsy, Epileptic Hemiplegia, Erb-Duchenne andDejerine-Klumpke Palsies, Erb's Palsy, Essential Tremor, ExtrapontineMyelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, FamilialDysautonomia, Familial Hemangioma, Familial Idiopathic Basal GangliaCalcification, Familial Periodic Paralyses, Familial Spastic Paralysis,Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, FisherSyndrome, Floppy Infant Syndrome, Foot prop, Friedreich's Ataxia,Frontotemporal Dementia, Gangliosidoses, Gaucher's Disease, Gerstmann'sSyndrome, Gerstmann-Straussler-Scheinker Disease, Giant AxonalNeuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, GloboidCell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen StorageDisease, Guillain-Barré Syndrome, Hallervorden-Spatz Disease, HeadInjury, Headache, Hemicrania Continua. Hemifacial Spasm, HemiplegiaAlterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia,Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes ZosterOticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly,HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease,Hydranencephaly, Hydrocephalus, Hydrocephalus—Normal Pressure,Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia,Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti, Infantile Hypotonia, Infantile NeuroaxonalDystrophy, Infantile Phytanic Acid Storage Disease, Infantile RefsumDisease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly,Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension,Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy'sDisease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-FeilSyndrome, Klippel-Trenaunay Syndrome (KTS), Klliver-Bucy Syndrome,Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg-WelanderDisease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-KleffnerSyndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral MedullarySyndrome, Learning Disabilities, Leigh's Disease, Lennox-GastautSyndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-CritchleySyndrome, Lewy Body Dementia, Lipid Storage Diseases, LipoidProteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease,Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications,Machado-Joseph Disease, Macrencephaly, Megalencephaly,Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis,Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy,Microcephaly, Migraine, Miller Fisher Syndrome, Mild CognitiveImpairment, Mini-Strokes, Mitochondrial Myopathies, Moebius Syndrome,Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease,Mucolipidoses, Mucopolysaccharidoses, Multifocal Motor Neuropathy,Multi-Infarct Dementia, Multiple Sclerosis, Multiple System Atrophy,Multiple System Atrophy with Orthostatic Hypotension, MuscularDystrophy, Myasthenia—Congenital, Myasthenia Gravis, MyelinoclasticDiffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy, Myopathy—Congenital, Myopathy-Thyrotoxic, Myotonia, MyotoniaCongenita, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Complications Of LymeDisease, Neurological Consequences of Cytomegalovirus Infection,Neurological Manifestations of Pompe Disease, Neurological Sequelae OfLupus, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,Normal Pressure Hydrocephalus, Occipital Neuralgia, Ohtahara Syndrome,Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, OrthostaticHypotension, O'Sullivan-McLeod Syndrome, Overuse Syndrome, Pain—Chronic,Pantothenate Kinase-Associated Neurodegeneration, ParaneoplasticSyndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis,Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, PenaShokeir II Syndrome, Perineural Cysts, Periodic Paralyses, PeripheralNeuropathy, Periventricular Leukomalacia, Persistent Vegetative State,Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick'sDisease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors,Polymyositis, Pompe Disease, Porencephaly, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Post-Polio Syndrome, PosturalHypotension, Postural Orthostatic Tachycardia Syndrome, PosturalTachycardia Syndrome, Primary Dentatum Atrophy, Primary LateralSclerosis, Primary Progressive Aphasia, Prion Diseases, ProgressiveHemifacial Atrophy, Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Prosopagnosia, Pseudotumor Cerebri, Ramsay HuntSyndrome I (formerly known as dyssynergia cerebellaris myoclonica,dyssynergia cerebellaris progressiva, dentatorubral degeneration, orRamsey Hunt cerebellar syndrome), Ramsay Hunt Syndrome II (formerlyknown as herpes zoster oticus), Rasmussen's Encephalitis, ReflexSympathetic Dystrophy Syndrome, Refsum Disease, Refsum.Disease—Infantile, Repetitive Motion Disorders, Repetitive StressInjuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, RettSyndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome,Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease,Sandhoff Disease, Schilder's Disease, Schizencephaly, SeitelbergerDisease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia,Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome,Shingles, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea, SleepingSickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal CordInfarction, Spinal Cord Injury, Spinal Cord Tumors, Spiral MuscularAtrophy, Spinocerebellar Atrophy, Spinocerebellar Degeneration,Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome,Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, SubacuteSclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy,SUNCT Headache, Swallowing Disorders, Sydenham Chorea, Syncope,Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, SystemicLupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts,Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome,Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, TicDouloureux, Todd's Paralysis, Tourette Syndrome, Transient IschemicAttack, Transmissible Spongiform Encephalopathies, Transverse Myelitis,Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical SpasticParaparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular ErectileTumor, Vasculitis Syndromes of the Central and Peripheral NervousSystems, Von Economo's Disease, Von Hippel-Lindau Disease (VHL), VonRecklinghausen's Disease, Wallenberg's Syndrome, Werdnig-HoffmanDisease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple'sDisease, Williams Syndrome, Wilson's Disease, Wolman's Disease, X-LinkedSpinal and Bulbar Muscular Atrophy, Zellweger Syndrome, optic neuritis,Chronic fatigue syndrome, fibromialgia, psychiataic diseases such asmood disorders, major depression, bipolar syndrome, psycosis,eschizophrenia, obsessive-compulsive-syndrome, etc., Toxic or drug abusediseases such as alcoholism and drug abuse, Encephalopathy like hepaticencephalopathy.

Psychiatric disorders, which may be the subject of prevention and/ortreatment according to the present invention include those listed by theDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition(DSM-IV) published by the American Psychiatric Association, and coversall mental health disorders for both children and adults. In particular,psychiatric disorders include a disorder selected from Acute StressDisorder; Adjustment Disorder Unspecified; Adjustment Disorder withAnxiety; Adjustment Disorder with Depressed Mood; Adjustment Disorderwith Disturbance of Conduct; Adjustment Disorder with Mixed Anxiety andDepressed Mood; Adjustment Disorder with Mixed Disturbance of Emotionsand Conduct; Agoraphobia without History of Panic Disorder; AnorexiaNervosa; Antisocial Personality Disorder; Anxiety Disorder Due toMedical Condition; Anxiety Disorder, NOS; Avoidant Personality Disorder;Bipolar Disorder NOS; Bipolar I Disorder, Most Recent Episode Depressed,In Full Remission; Bipolar I Disorder, Most Recent Episode Depressed, InPartial Remission; Bipolar I Disorder, Most Recent Episode Depressed,Mild; Bipolar I Disorder, Most Recent Episode Depressed, Moderate;Bipolar I Disorder, Most Recent Episode Depressed, Severe With PsychoticFeatures; Bipolar I Disorder, Most Recent Episode Depressed, SevereWithout Psychotic Features; Bipolar I Disorder, Most Recent EpisodeDepressed, Unspecified; Bipolar I Disorder, Most Recent Episode Manic,In Full Remission; Bipolar I Disorder, Most Recent Episode Manic, InPartial Remission; Bipolar I Disorder, Most Recent Episode Manic, Mild;Bipolar I Disorder, Most Recent Episode Manic, Moderate; Bipolar IDisorder, Most Recent Episode Manic, Severe With Psychotic Features;Bipolar I Disorder, Most Recent Episode Manic, Severe Without PsychoticFeatures; Bipolar I Disorder, Most Recent Episode Manic, Unspecified;Bipolar I Disorder, Most Recent Episode Mixed, In Full Remission;Bipolar I Disorder, Most Recent Episode Mixed, In Partial Remission;Bipolar I Disorder, Most Recent Episode Mixed, Mild; Bipolar I Disorder,Most Recent Episode Mixed, Moderate; Bipolar I Disorder, Most RecentEpisode Mixed, Severe With Psychotic Features; Bipolar I Disorder, MostRecent Episode Mixed, Severe Without Psychotic Features; Bipolar IDisorder, Most Recent Episode Mixed, Unspecified; Bipolar I Disorder,Most Recent Episode Unspecified; Bipolar I Disorder, Most Recent EpisodeHypomanic; Bipolar I Disorder, Single Manic Episode, In Full Remission;Bipolar I Disorder, Single Manic Episode, In Partial Remission; BipolarI Disorder, Single Manic Episode, Mild; Bipolar I Disorder, Single ManicEpisode, Moderate; Bipolar I Disorder, Single Manic Episode, Severe WithPsychotic Features; Bipolar I Disorder, Single Manic Episode, SevereWithout Psychotic Features; Bipolar I Disorder, Single Manic Episode,Unspecified; Bipolar II Disorder; Body Dysmorphic Disorder; BorderlinePersonality Disorder; Breathing-Related Sleep Disorder; Brief PsychoticDisorder; Bulimia Nervosa; Circadian Rhythm Sleep Disorder; ConversionDisorder; Cyclothymic Disorder; Delusional Disorder; DependentPersonality Disorder; Depersonalization Disorder; Depressive DisorderNOS; Dissociative Amnesia; Dissociative Disorder NOS; DissociativeFugue; Dissociative Identity Disorder; Dyspareunia; Dyssomnia NOS;Dyssomnia Related to Another Disorder; Dysthymic Disorder; EatingDisorder NOS; Exhibitionism; Female Dyspareunia Due to MedicalCondition; Female Hypoactive Sexual Desire Disorder Due to MedicalCondition; Female Orgasmic Disorder; Female Sexual Arousal Disorder;Fetishism; Frotteurism; Gender Identity Disorder in Adolescents orAdults; Gender Identity Disorder in Children; Gender Identity DisorderNOS; Generalized Anxiety Disorder; Histrionic Personality Disorder;Hypoactive Sexual Desire Disorder; Hypochondriasis; Impulse-ControlDisorder NOS; Insomnia Related to Another Disorder; IntermittentExplosive Disorder; Kleptomania; Major Depressive Disorder, Recurrent,In Full Remission; Major Depressive Disorder, Recurrent, In PartialRemission; Major Depressive Disorder, Recurrent, Mild; Major DepressiveDisorder, Recurrent, Moderate; Major Depressive Disorder, Recurrent,Severe With Psychotic Features; Major Depressive Disorder, Recurrent,Severe Without Psychotic Features; Major Depressive Disorder, Recurrent,Unspecified; Major Depressive Disorder, Single Episode, In FullRemission; Major Depressive Disorder, Single Episode, In PartialRemission; Major Depressive Disorder, Single Episode, Mild; MajorDepressive Disorder, Single Episode, Moderate; Major DepressiveDisorder, Single Episode, Severe With Psychotic Features; MajorDepressive Disorder, Single Episode, Severe Without Psychotic Features;Major Depressive Disorder, Single Episode, Unspecified; Male DyspareuniaDue to Medical Condition; Male Erectile Disorder; Male Erectile DisorderDue to Medical Condition; Male Hypoactive Sexual Desire Disorder Due toMedical Condition; Male Orgasmic Disorder; Mood Disorder Due to MedicalCondition; Narcissistic Personality Disorder; Narcolepsy; NightmareDisorder; Obsessive Compulsive Disorder; Obsessive-CompulsivePersonality Disorder; Other Female Sexual Dysfunction Due to MedicalCondition; Other Male Sexual Dysfunction Due to Medical Condition; PainDisorder Associated with both Psychological Factors and MedicalConditions; Pain Disorder Associated with Psychological Features; PanicDisorder with Agoraphobia; Panic Disorder without Agoraphobia; ParanoidPersonality Disorder; Paraphilia, NOS; Parasomnia NOS; PathologicalGambling; Pedophilia; Personality Disorder NOS; Posttraumatic StressDisorder; Premature Ejaculation; Primary Hypersomnia; Primary Insomnia;Psychotic Disorder Due to Medical Condition, with Delusions; PsychoticDisorder Due to Medical Condition, with Hallucinations; PsychoticDisorder, NOS; Pyromania; Schizoaffective Disorder; Schizoid PersonalityDisorder; Schizophrenia, Catatonic Type; Schizophrenia, DisorganizedType; Schizophrenia, Paranoid Type; Schizophrenia, Residual Type;Schizophrenia, Undifferentiated Type; Schizophreniform Disorder;Schizotypal Personality Disorder; Sexual Aversion Disorder; SexualDisorder NOS; Sexual Dysfunction NOS; Sexual Masochism; Sexual Sadism;Shared Psychotic Disorder; Sleep Disorder Due to A Medical Condition,Hypersomnia Type; Sleep Disorder Due to A Medical Condition, InsomniaType; Sleep Disorder Due to A Medical Condition, Mixed Type; SleepDisorder Due to A Medical Condition, Parasomnia Type; Sleep TerrorDisorder; Sleepwalking Disorder; Social Phobia; Somatization Disorder;Somatoform Disorder NOS; Specific Phobia; Transvestic Fetishism;Trichotillomania; Undifferentiated Somatoform Disorder; Vaginismus; andVoyeurism.

Preferably, the compounds of any of Formulae I-V, and pharmaceuticallyacceptable salts and prodrugs thereof, can be used in the treatment ofdiseases wherein NGF or other neurotrophins have been proven effectivein the state of the art, either in vivo or in vitro, due to theirimproving effects on cell differentiation and cell survival, througheither TrkA, TrkB and/or p75 pathways. Therefore, the compounds coveredin the present invention can be used in the treatment of neurologicaldiseases selected among: neurodegenerative disorders, such asamyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, Friedreich's ataxia, Huntington's disease, Dementia with Lewybodies, spinal muscular atrophy; nerve inflammation, such as multiplesclerosis and neuromyelitis optica; major depressive disorder;schizophrenia; glaucoma; or peripheral neuropathies, such as diabetic orAIDS neuropathy. Moreover, the compounds of the invention can also beindicated for treatment of cancer, by modulating NGF celldifferentiation activity and stopping cell proliferation. Among thecancer types in which NGF has been proven effective in the state of theart, either in vivo or in vitro, due to improving effects on celldifferentiation and cell survival, through either TrkA and/or p75pathways, the following may be cited: glioblastoma, astrocytoma,meduloblastoma, neurinoma, neuroblastoma, meningioma, colon cancer,pancreatic cancer, breast cancer, prostate cancer, leukemia, acutelymphocytic leukemia, osteosarcoma, hepatocellular carcinoma, ovariancarcinoma, lung adenocarcinoma or esophagic carcinoma.

In one embodiment of the present invention, in the various uses andmethods of neuroprotection or of prevention or treatment of nerve celldeath or damage, the compounds of any of Formulae I-V, andpharmaceutically acceptable salts and prodrugs thereof, are administeredto a healthy subject, preferably a healthy subject older than 18 yearsold, more preferably a healthy subject older than 45 years old, evenmore preferably a healthy subject older than 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 years old.

The term “healthy subject” is meant to comprise its plain meaning aswell as those subjects that may suffer from one or more pathologicalconditions other than a neurological or psychiatric disease.

The neuroprotective properties of the compounds of any of Formulae I-V,and pharmaceutically salts and prodrugs thereof, have as a consequencethe partial or full prevention or treatment of the various disorders inthe nervous system functions caused by the neuronal cell death ordamage. Therefore, the present invention further relates to the use ofthe compounds of any of Formulae I-V, and pharmaceutically acceptablesalts and prodrugs thereof, as active ingredients in the manufacture ofa medicament for the prevention or treatment of a neurological orpsychiatric disease. In other words, the present invention also relatesto the compounds of any of Formulae I-V, and pharmaceutically acceptablesalts and prodrugs thereof, for use in the prevention or treatment of aneurological or psychiatric disease. Similarly, the present inventionalso relates to a method of prevention or treatment of a neurological orpsychiatric disease comprising administering to a subject in needthereof an effective amount of the compound of any of Formulae I-V, or apharmaceutically acceptable salt or prodrug thereof. The neurological orpsychiatric disease may be any one from those listed above.

Preferably, the neurological or psychiatric disease is selected fromneurodegenerative disorders, inflammation and certain types of cancers,multiple sclerosis, neuromyelitis optica amyotrophic lateral sclerosis(ALS), Parkinson's disease, Alzheimer's disease, Friedreich's ataxia,Huntington's disease, Dementia with Lewy bodies, spinal muscularatrophy, major depressive disorder, schizophrenia, glaucoma orperipheral neuropathies (diabetic or AIDS neuropathy).

Another goal of present invention is the use of the compounds of any ofFormulae I-V, and pharmaceutically acceptable salts and prodrugsthereof, as neuroenhancing drugs or the use for manufacturingneuroenhancing drugs.

Neuroenhancing drugs include those that improve learning and memory,attention, mood, communicative skills and sexual performance. Examplesof neuroenhancing drugs are those that target long-term synapticpotentiation (LTP) or long-term depression (LTD), modulation of calciumchannels, or the cAMP response element-binding (CREB) protein. cAMP isthe acronym for cyclic adenosine monophosphate. Particular examples ofneuroenhancing drugs are phosphodiesterase inhibitors like rolipram;donepezil; agonists of the NMDA glutamate receptor like D-cycloserine;ampakines; modafinil; methylphenidate.

A pharmaceutical composition of the present invention can beadministered by any means that achieves its intended purpose. Forexample, the administration can be oral, parenteral, subcutaneous,intravenous, instramuscular, intraperinoneal, transdermal, intranasal,transmucosal, rectal, or buccal route. In one embodiment, thepharmaceutical composition is administered orally.

The compounds of any of Formulae I-V, and pharmaceutically acceptablesalts and prodrugs thereof, may be formulated into variouspharmaceutical forms for administration purposes. As appropriatecompositions there may be cited all compositions usually employed forsystemically administering drugs, for example any solid (e.g. tablets,capsules, granules, etc.) or liquid composition (e.g. solutions,suspensions, emulsions, etc). To prepare the pharmaceutical compositionsof the compounds of any of Formulae I-V, an effective amount of thecompound of any of Formulae I-V, optionally in a salt form or a prodrug,as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirable inunitary dosage form suitable, particularly, for administration orally,rectally, percutaneously, intrathecal, intravenous or by parenteralinjection. For example, in preparing the compositions in oral dosageform, any of the usual pharmaceutical media may be employed such as, forexample, water, glycols, oils, alcohols and the like in the case of oralliquid preparations such as suspensions, syrups, elixirs, emulsions andsolutions; or solid carriers such as starches, sugars, kaolin,lubricants, binders, disintegrating agents and the like in the case ofpowders, pills, capsules, and tablets. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit forms, in which case solid pharmaceutical carriers areobviously employed. For parenteral compositions, the carrier usuallycomprises sterile water, at least in large part, though otheringredients, for example, to aid solubility, may be included. Injectablesolutions, for example, may be prepared in which the carrier comprisessaline solution, glucose solution or a mixture of saline and glucosesolution. Injectable suspensions may also be prepared in which caseappropriate liquid carriers, suspending agents and the like may beemployed. Also included are solid form preparations, which are intendedto be converted, shortly before use, to liquid form preparations. In thecompositions suitable for percutaneous administration, the carrieroptionally comprises a penetration enhancing agent or a suitable wettingagent, or both, optionally combined with suitable additives of anynature in minor proportions, which additives do not introduce asignificant deleterious effect on the skin. A review of the differentpharmaceutical forms for drug administration and their preparation maybe found in the book “Tratado de Farmacia Galénica”, de C. Faulí iTrillo, 10th Edition, 1993, Luzan 5, S.A. de Ediciones.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills,suppositories, powder packets, wafers, injectable solutions orsuspensions and the like, and segregated multiples thereof.

The compositions in accordance with this invention, including unitdosage forms, may contain the active ingredient in an amount that is inthe range of about 0.1% to 70%, or about 0.5% to 50%, or about 1% to25%, or about 5% to 20%, the remainder comprising the carrier, whereinthe foregoing percentages are w/w versus the total weight of thecomposition or dosage form.

The dose of the compound of any of Formulae I-V, its pharmaceuticallyacceptable salt or prodrug thereof, to be administered depends on theindividual case and, as customary, is to be adapted to the conditions ofthe individual case for an optimum effect. Thus it depends, of course,on the frequency of administration and on the potency and duration ofaction of the compound employed in each case for therapy or prophylaxis,but also on the nature and severity of the disease and symptoms, and onthe sex, age, weight co-medication and individual responsiveness of thesubject to be treated and on whether the therapy is acute orprophylactic. Doses may be adapted in function of weight and forpediatric applications. Daily doses may be administered q.d. or inmultiple quantities such as b.i.d., t.i.d. or q.i.d.

Synthesis of Compounds

The compounds of the present invention can be prepared using methodsknown to those skilled in the art in view of this disclosure. Forexample, compounds of the present invention can be prepared as describedin Masip, et al., 2005.

Testing of Compounds

In addition to the tests described in the Examples, the compounds of thepresent invention can be tested for Alzheimer disease in vitro model asfollows: The human neuroblastoma cell line SH-SY5Y is used to study theneuroprotective effect of the tested compound in Alzheimer disease. Thecells are pre-treated for 3 hours with the tested compound at differentconcentrations (20 ng/ml, 100 ng/ml, 2 μg/ml, 20 μg/ml and 50 μg/ml)with the tested compound (100 ng/ml). Then Amiloid beta fibrils (100 μM)is added and incubated for 24 hrs. The number of surviving cells isdetermined the day after by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay.

Induction of TrkA, IκBa and SAPK/JNK phosphorylation by the compounds ofthe present invention can be tested as follows. Activation of TrkA isthe first event in the signalling cascade leading to differentiation andsurvival of NGF responsive neurons and PC12 cells (Greene and Tischler,1976; Chao, 2003; Huang and Reichardt, 2003). To evaluate whether theneurotrophic activity of NGF-like peptoids is mediated by theinteraction with TrkA receptor, their capacity to induce TrkAphosphorylation in PC12 cells can be analyzed. The activity of peptoidscan be tested in the range of concentrations that were effective on PC12cells differentiation.

Western blot analysis. Subconfluent cells are grown overnight in mediumcontaining 2% FBS and 1% HS and stimulated with 100 ng/ml NGF or theNGF-like small chemicals for the indicated time points. Cells are thenwashed with cold phosphate-buffered saline (PBS) and briefly sonicatedin SDS sample buffer (containing β-mercaptoethanol and 2 mM PMSF).Lysates (200 μg of total proteins) are separated on SDS-PAGE, andtransferred to nitrocellulose (Whatman, Dassel, Germany). After blockingwith 5% nonfat milk in TBST buffer (10 mM Tris pH 7.5; 150 mM NaCl/0.2%Tween 20), blots are probed overnight at 4° C. with anti-p-TrkA (Tyr490) antibody (1:1000), anti TrkA antibody (1:1000), (Ser 32/36)(5A5)mouse antibody (1/1000), anti IκBα (L35A5) mouse antibody (1:1000),anti-p-SAPK/JNK (Thr183/Tyr185) antibody (1:1000) or anti-SAPK/JNKantibody (1/1000)(all of them from Cell Signaling), followed byincubation with HRP-conjugated IgG (Jackson ImmunoResearch) for 1 h atroom temperature (RT). Detection of phosphorylated species is performedby using the enhanced chemiluminescence (ECL) system (GE HealthcareBio-Sciences, Piscataway, N.J.).

To investigate whether the compounds of the present invention activatep75, the activation of the NF-κB pathway (Bonizzi G, Karin M. 2004) andthe SAPK/JNK pathway (cell death) in PC12 and RN22 cell lines can beanalyzed. RN22 cells express high levels of the p75 receptor message andprotein, whereas TrkA expression is undetectable (Gentry et al., 2000).NF-κB is functionally active as a transcriptional regulator in a dimericform consisting of homo- or heterodimers, the prototypic NF-κB dimmerconsisting of the p65 and p50 subunits. The activation of NF-κB occursprimarily through the degradation of the IκB proteins, a family ofinhibitory proteins bound to NF-κB dimers. In response to activatingstimuli, the inhibitory proteins are phosphorylated, which targets themfor ubiquitination and subsequent degradation. One member of theinhibitory family, IκBα, is degraded in response to the majority of theNF-κB activators (Ghosh et al., 1998). To examine NF-κB activation inresponse to NGF and the different selected peptoids, westerns blots oftotal cell extracts from RN22 and PC12 cells treated with NGF and thepeptoids for varying times are probed with an antibodyanti-phospho-IκBα.

In several neuronal systems, JNK activation has been causally linkedwith the induction of programmed cell death (Bhakar et al., 2003). Inculture, NGF signalling through p75 led to activation of both NF-Kb andJNK, resulting ultimately in programmed cell death (Yoon et al., 1998).To examine the activity of JNK in RN22 cells western blots can be donewith antibody anti-phospho-SAPK/JNK to assess activation of the pathway.

Effect of neurotrophin peptidomimetics (e.g., NGF-mimetic peptoids) inencephalomyelitis autoimmune experimental (EAE) can be assessed asfollows:

Animals, Experimental Autoimmune Encephalomyelitis Induction, andTreatment. Trials are approved by the University of Barcelona Committeeon Animal Care. Female C57BL/6 mice from Harlan (8-12 weeks old) areimmunized subcutaneously in both hind pads with 300 μg of myelinoligodendrocyte glycoprotein (MOG) peptide 35-55 (Spikem, Firenze)emulsified with 50 μg of Mycobacterium tuberculosis (H37Ra strain;Difco, Detroit, Mich.) in incomplete Freund's adjuvant (IFA) aspreviously described (Palacios et al. 2007). Mice are intraperitoneallyinjected with Pertussis toxin (Sigma)(500 ng) at the time ofimmunization and 2 days later. Animals are weighted and inspected forclinical signs of disease on a daily basis by a blinded observer.Disease severity of EAE is assessed according to the following scale:0=normal; 0.5=mild limp tail; 1=limp tail; 2=mild parapesis of the hindlimbs, unsteady gait; 3=moderate parapesis, voluntary movements stillpossible; 4=paraplegia or tetraparesis; 5=moribund state. Data shown forthe clinical studies are representative of two independent experimentsperformed with the indicated number of animals (Moreno et al., 2006).

The test compounds are prepared in water with 5% DMSO. Animals aretreated with the test compound (25, 50 and 100 mg/kg) or placebo(water+5% DMSO) through daily intraperitoneal injection starting afterimmunization. At the end of the study, mice are anesthetized andperfused intracardially with 4% of paraformaldehyde in 0.1M phosphatebuffer (pH 7.6). Brains, spinal cords and spleens are dissected andeither fixed or frozen until use. Serum is obtained from all animalsincluded in the study, and transaminases levels are measured.

In order to evaluate the effects of the neurotrophin peptidomimetics(e.g., the NGF-like peptoids) “in vivo”, the effect of the neurotrophinpeptidomimetics (e.g., the NGF-mimetic peptoids) in the animal model ofMS can be studied. C57BL/6 mice immunized with MOG35-55 peptide aretreated daily with a test compound intraperitoneally from day 0 to day25 at a different concentrations of the compound. Effect of thecompounds of the present invention in CNS and peripheral inflammationcan be tested as follows.

Real-time Quantitative Polymerase Chain Reaction. Brains and spinalcords from mice obtained at the time of death are homogenized in RNAlysis buffer. Total RNA is extracted using the RNeasy Mini Kit (Qiagen,Chatwworth, Calif.) isolation system, including DNase treat nent usingthe RNase-Free DNase Set (Quiagen). Total RNA (35 μg) is reversetranscribed using the Reverse Transcription System (High Capacity cDNAArchive Kit; Applied Biosystems, Foster City, Calif.). The real timereaction is conducted at 25° C. for 10 minutes, followed by 37° C. for 2hours, and finally stored at 4° C. Primers and target-specificfluorescence-labeled TaqMan probes can be purchased from AppliedBiosystems (TaqMan Gene Expression assays). For example, the TaqManUniversal Master Mix (Applied biosystems) can be used. Amplification ofcomplementary DNA is performed on a DNA Engine Opticon 2 Real-TimeSystem (MJ Research, Watertown, Mass.) using 0.9 μM for each primer and0.25 μM for the probe and 20 ng complementary DNA. The reactionconditions are an initial 2 minutes at 50° C., followed by 10 minutes at95° C. and 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Eachsample is run in triplicate, and in each plate the target and theendogenous control are amplified in different wells. The expression ofthe gene tested is quantified relative to the level of the housekeepinggene 18rRNA (Palacios et al., 2008).

Immunohistochemistry. Histological evaluation is done onparaformaldehyde-fixed, paraffin-embedded sections of brain and spinalcord. Sections 10 μm thick) are stained with hematoxylin and Luxol FastBlue to assess inflammation and demyelination, Semiquantitativehistological evaluation for inflammation and demyelination is conductedand scored blindly using the following scale: 0=normal; 1=1 to 3/sectionperivascular cuffs with minimal demyelination; 2=3 to 10 perivascularcuffs/section accompanied by moderate demyelination; 3=wide-spreadperivascular cuffing, extensive demyelination with large confluentlesions (Villoslada et al., 2001).

Immunohistochemical procedures are performed on 10 μm paraffin-embeddedsections of brain and spinal cord as described previously (Villoslada etal., 2001). Primary antibodies are added at concentrations of 1/1000 forMCA500 (rat anti-mouse CD3 from Serotec) and 1/500 for MCA1107 (ratanti-mouse CD4 from Serotec). Specificity of the immunoreaction isdetermined by incubating sections without the primary antibodies orusing the corresponding isotype controls which yielded noimmunoreactivity.

Proliferation assay. Splenocytes from naïve, non immunized C57BL/6 miceare obtained for in vitro assessment of the effect of the test compoundin cell proliferation. Splenocyte proliferation assay is performed asdescribed previously (Martinez-Forero et al., 2008).

To assess the effect of the test compound in the peripheral immuneresponse, the proliferative response against the immunizing antigen(MOG) in splenocytes of naïve animals and the cytokine profile in spleencells from placebo and treated animals are evaluated. Gene expression ofinterleukin2 (IL2), Interferon γ (IFNγ), tumor necrosis factor α (TNFα),inducible nitric oxide synthase (iNOS) and interleukin 10 (IL 10) can beinvestigated by quantitative reverse transcriptase PCR at the end of theexperiment in splenocytes from placebo and treated animals.

Statistical analysis. Statistical analyses can be performed with thetwo-tailed Mann-Whitney U test for comparing EAE scores, chi 2 test forcomparing disease incidence and Kaplan-Meier curves for differences inday of onset of EAE. p values less than 0.05 are considered to indicatea significant difference. The statistical evaluation is conducted usingthe SPSS16.0 statistical program (SPSS, Chicago, Ill.).

The following examples are illustrative, but not limiting, of thecompounds, compositions and methods of the present invention. Suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art in view of this disclosure arewithin the spirit and scope of the invention.

EXAMPLES Example 1 Design of NGF Agonists by Combinatorial Chemistry

General. Solvents, amines and other reagents were purchased fromcommercial suppliers and used without further purification. Reactionscarried out under microwave irradiation were conducted in a 100 mL roundbottomed flask equipped with a Dimroth condenser. The flask wasintroduced in the monomode cavity of a CEM Model Discover apparatus. TheNMR spectra were recorded on a Varian Inova 500 apparatus (¹H NMR, 500MHz; ¹³C NMR, 125 MHz) and on a Unity 300 apparatus (¹H NMR, 300 MHz;¹³C NMR, 75 MHz). When appropriate, the assignment of ¹H and ¹³C NMRpeaks for compounds were confirmed by gDQCOSY and gHSQC experiments. Theoccurrence of different conformers led to highly complex spectra; theabsorptions given below are referred to the major conformer present inthe sample. The RP-HPLC analyses were performed with a Hewlett PackardSeries 1100 (UV detector 1315A) modular system using a reverse-phaseKromasil 100 C8 (25×0.46 cm, 5 μm) column, with CH₃CN-buffer ammoniumformate (20 mM, pH=5.0) mixtures at 1 mL/min as mobile phase andmonitoring at 220 nm. Semi-preparative RP-HPLC was performed with aWaters (Milford, Mass., U.S.A.) system. High resolution mass spectra(HRMS-FAB) were carried out at the IQAC—Instituto de Quimica Avanzada deCataluña—(Spain).

Synthesis of individual peptoids. The synthesis of individual peptoidsG79, G80, and G81 for the in vitro and in vivo assays was carried outfollowing the substructure procedure reported by the group of Zuckermannwith some modifications (Zuckermann et al, 1992)

The synthesis was carried out on a 1% cross-linked polysterene resinbearing the Fluorenylmethoxycarbonyl (Fmoc)-protected Rink amide linkerAM RAM (0.79 mmol/g, Rapp Polymer; Germany). A suspension of 4 g ofresin in 50 mL DMF was placed in a 100 mL round bottomed flask providedwith a magnetic stirrer. The suspension was stirred for 5 min at 20° C.,the solvent was removed by filtration through a 60 mL polypropylenesyringe provided with a polyethylene porous plaque. Then, resin wastransferred again to the reaction flask.

Fmoc deprotection. A solution of 60 mL of 20% piperidine in DMF wasadded to the roundbottomed flask containing the resin. The mixture wasallowed to react under microwave activation for 5 min at 60° C. and 150W. The resin was drained on the 60 mL syringe and washed with 40 mL DMF.The treatment was carried out in duplicate. Then, the resin was filteredand washed with DMF (3×40 mL), iPrOH (3×40 mL), and CH₂Cl₂ (3×40 mL).Finally, it was drained for 2 min and transferred to the reaction flask.The deprotection was monitored by using the TNBS test (red colour aspositive).

2. First acylation. The resin was treated with a solution of 5equivalents of bromoacetic acid (2.2 g, 15.8 mmol) and 5 equivalents ofN,N-diisopropylcarbodiimide (2.5 mL, 15.8 mmol) in 50 mL of DMF. Theacylation was conducted under microwave irradiation (5 min, 60° C., 150W). Then, the resin was filtered using the syringe, and washed with 40mL of DMF. The reaction was carried out in duplicate. Afterwards, theresin was filtered and washed with DMF (3×40 mL), iPrOH (3×40 mL), andCH₂Cl₂ (4×10 mL) Next, it was drained for 2 min and the absence ofprimary amine was evaluated by TNBS test.

3. First Amination coupling. A suspension of the acylated resin in 50 mLDMF was treated with the suitable primary amine according to the finalcompound: 5 equivalents (2.2 mL, 15.8 mmol) of 2.2 mL1-(3-aminopropyl)-2-pyrrolidinone, or 5 equivalents (3.2 g, 15.8 mmol)of 4-(2-aminoethyl)benzenesulfonamide. The reaction was conducted undermicrowave irradiation (7 min, 80° C., 150 W). The reaction was carriedout in duplicate, washing and draining the resin through the syringebetween the treatments. Finally, the resin was drained for 2 min andtransferred to the flask. The incorporation of the amine was confirmedby the chloranil test (green colour for secondary amines).

4. Second and third acylation steps. They were carried out similarly tothe first acylation step. In this case, two acylation treatments wereenough to complete the reaction.

5. Second and third amination coupling steps. They were carried outsimilarly to the first amination step, bu using the correspondingprimary amines. Thus, 5 equivalents (1.8 ml, 15.8 mmol) of2-methyl-1-propanamine were used for the second amination coupling. Forthe third amination, 5 equivalents (2.0 mL, 15.8 mmol) of2-(2-fluorophenyl)ethanamine or 5 equivalents (2.0 mL, 15.8 mmol) of2-(1-pyrrolidinyl)ethanamine were used as according to the compositionof the corresponding peptoid.

6. Cleavage. After draining the resin, it was divided into 4 aliquotesand each one was treated with 20 mL of the cleavage cocktail (60:40:2(v/v/v) TFA/CH₂Cl₂/H₂O). The mixtures were stirred for 30 min at 20° C.and filtered through a 10 mL polypropylene syringes provided with apolyethylene porous plaque. The filtrates were collected in a 250 mLflask and solvents were removed under reduced pressure. Finally, theyellow oil residue that was obtained for the case of each peptoid wasredissolved in H₂O/ACN mixture and lyophilized to give 1.25-1.80 g ofthe expected crude peptoid in purities higher than 85% by HPLC.

7. Purification and chemical characterization. Compounds were purifiedby semipreparative HPLC using a Waters PrePack®-C₁₈ (47×300 mm, 15-μm,)column, eluting with CH₃CN/H₂O mixtures containing 0.1% TFA as mobilephases, and a flow rate of at 60 mL/min. Final compounds were obtainedin purities higher to 98% by HPLC. Quantities obtained were: 10.64 mg ofG79 (99% purity; HPLC), 7.55 mg of G80 (99% purity; HPLC), and 12.68 mgof G81 (99% purity; HPLC).

[N-(2-(2′-Fluorophenyl)ethyl)glycyl]-[N-(2-methylpropyl)glycyl]-N-[3-(2-oxopyrrolidinyl)propyl]glycinamide(G79). Chemical Formula: C₂₅H₃₈FN₅O₄; MW 491, 5987.

[N-(2-(2′-Fluorophenyl)ethyl)glycyl]-[N-(2-methylpropyl)glycyl]-N-[2-(4′-sulfamoylphenyl)ethyl]glycinamide(G80). Chemical Formula: C₂₆H₃₆FN₅O₅S; MW 549, 658.

[N-(2-(1-Pyrrolidinyl)ethyl)glycyl]-[N-(2-methylpropyl)glycyl]-N-[2-(4′-sulfamoylphenyl)ethyl]glycinamide(G81). Chemical Formula: C₂₄H₄₀N₆O₅S; MW 524, 6766.

Example 2 Dose Response Differentiation Assay in PC12 Cell Line

Cell culture: Stock of PC12 cultures were maintained in Ham's F12 mediumsupplemented with 2.5% fetal bovine serum (FBS), 15% of horse serum (HS)and 1% penicillin/streptomycin. The cell culture was maintained in 5%CO₂ at 37° C.

PC12 cell differentiation was assessed by treating the cells with allthe test compounds (G79, G80, and G81) at different testingconcentration (2-20-100 ng/ml and 2-20-50 μg/ml). The cells were firstplated into collagen-coated 24-wells plates with 2.5% FBS in the mediumto be de-differentiated. After 72 hrs, the test compounds were added andNGF (Sigma Aldrich; 100 ng/ml) as positive control of differentiation.The number of differentiated cells, with neurite processes greater thantwo cell bodies in length, is counted after 3 days of treatment. Thecell count is done in three randomly selected fields with 100 cells.

Compounds G79, G80 and G81 induce the differentiation of PC12 cells(FIG. 1A-1D). The NGF positive control induce a differentiation of16.1±1.9 cells in a field of 100 cells. G79 induces significantdifferentiation, compared to control PC12 untreated cells, in a dosedependent manner (2-20-100 ng/ml and 2-20 μg/ml). The best workingconcentration is 20 μg/ml (number of cells: 11.3±2.68). At 50 μg/ml thenumber of differentiated cells decreases.

G80 induces good differentiation with very long neurites in PC12 cells.The best working concentration is 20 ng/ml. G81 induces significantdifferentiation, compared to control PC12 untreated cells, in a dosedependent manner until 100 ng/ml, that is the best working concentration(number of cells: 9.2±3.9). For higher concentration the number ofdifferentiated cells decreases. The percentage of differentiated cellsis calculated as relative to NGF induced differentiation (FIG. 1E).

Example 3 Survival Assays G79, G80 and G81 Neurotrophin PeptidomimeticsPromote Cell Survival

Cell culture. The rat schwannome cell line RN22 was cultured inDulbecco's modified Eagle's medium (DMEM) with 10% FBS and 1%penicillin/streptomycin. The cell culture was maintained in 5% CO₂ at37° C.

RN22 were plated into 24-wells plates at the concentration of 30.000cells/well in medium without serum. After 24 h, the test compounds G79,G80, and G81 were added at different concentrations (1-10-50 ng/ml and1-10 μg/ml) and NGF was used as positive control of survival. The cellswere so incubated for 2 hrs. Oxidative stress was induced with coppersulphate (CuSO4) at the final concentration of 150 μg/ml. Afterover-night incubation cell viability was determined by reading theabsorbance after adding MTT (Sigma Aldrich).

The percentage of surviving cells was calculated as relative to NGFinduced differentiation (FIG. 2). G79 increased cell viability at alltested concentrations (1-10-50 ng/ml and 1-10 μg/ml) with a best workingconcentration at 10 ng/ml (98.11% of cell viability, as % relative tothe no-stress control). G80 increased cell viability at all testedconcentrations (1-10-50 ng/ml and 1-10 μg/ml) with a best workingconcentration at 50 ng/ml (99.86% cell viability, as % relative to theno-stress control). G81 increased cell viability at all testedconcentrations (1-10-50 ng/ml and 1-10 μg/ml) with a best workingconcentration at 50 ng/ml (101.86% of cell viability, as % relative tothe no-stress control).

Example 4 Secretagogue Activity Assay G79, G80, and G81 do not InduceNGF Secretion

To assess if the tested compounds, G79, G80, and G81, act assecretagogues, inducing neurotrophic activity through the synthesis ofNGF, PC12 cells were plated into collagen-coated 24-wells plates withonly 2.5% FBS and after 72 hrs the cells were treated with the testedcompounds (each one at 100 ng/ml) together with antibody anti-NGF (1μg/ml, AbCam). After three days of treatment, the differentiation wasevaluated. The cell count was done in three randomly selected fieldswith 100 cells.

Treatment with G79, G80 or G81 together with antibody anti-NGF inducePC12 differentiation similarly to the differentiation induced by thetested compounds alone (FIG. 3A). Wile the number of differentiatedcells by NGF is decreased by adding anti-NGF antibody (12.8±6.3 vs20.7±7.1), the number of differentiated cells results to be quitesimilar comparing the treatment with the tested compounds alone with thetreatment with this compounds and anti-NGF antibody. The G79/anti-NGFtreatment obtained a number of differentiated cells for each fieldresults to be 18.8±4.9 vs 11.5±2.8 for the treatment with only G79; forG80/anti-NGF is 14.1±1.8 vs 15.1±6.3 for the treatment with only G80;for G81/anti-NGF is 15.5±4.1 vs 14.1±3.3. The percentage ofdifferentiated cells is calculated as relative to NGF induceddifferentiation (FIG. 3F).

Example 5 Synergistic Activity Assay

G79, G80, and G81 do not have a Synergistic Activity with NGF

To evaluate if the small molecules G79, G80, and G81 interfere with themaximal activity of NGF or lead to additive effects, PC12 cells wereplated into collagen-coated 24-wells plates with 2.5% FBS and treatedafter 72 h with the small chemicals (each one at 100 ng/ml) incombination with NGF at the concentration of 100 ng/ml. In the sameexperiment PC12 cells were treated with the small molecules or NGFalone, as controls. The number of differentiated cells was evaluatedafter three days of treatment. The cell count was done in three randomlyselected fields with 100 cells

The results are shown in FIG. 3B. The treatment with G79, G80 or G81molecules together with NGF decrease the activity of NGF, resulting intoa decreased number of differentiated cells in the combination treatmentNGF/G79-G80-G81. While the number of NGF differentiated cells in thisexperiment results to be 20±7.1, the number of differentiated cellsobtained by the treatment NGF/G79 results to be 11.3±2.6. With thetreatment NGF/G80 the number of differentiated cells obtained is 13±5.9.With the treatment NGF/G81 the number of differentiated cells obtainedis 13.3±3.8. Overall, these results rule out that G79, G80 or G81 mayhave additive, synergistic or antagonistic effect with NGF. Thepercentage of differentiated cells is calculated as relative to NGFinduced differentiation (FIG. 3G).

Example 6 Receptor Activation Assay

The hypothesis that G79, G80, and G81 can act through the binding to theextracellular portion of TrkA (binding site) and activate the tyrosinekinase receptor was tested by treating the PC12 cells with the smallchemicals in combination with the antibody anti-TrkA or the K252a, atyrosine kinase inhibitor. For this purpose PC12 cells were plated intocollagen-coated 24-wells plates with 2.5% FBS and after 72 hrs wereincubated with the small molecules alone or in combination withanti-TrkA antibody (AbCam, 1:2000) or K252a. NGF (100 ng/ml) was used ascontrol and added alone or in combination with anti-TrkA antibody orK252a. The number of differentiated cells was evaluated after three daysof treatment. The cell count was done in three randomly selected fieldswith 100 cells. The results are shown in FIG. 3C.

Treatment with G79, G80 and G81 molecules together with antibodyanti-TrkA did not inhibit the differentiation of PC12 cells. While thenumber of differentiated cells by NGF is decreased by adding antiTrkAantibody (12.8±6.3 vs 20.7±7.1), the number of differentiated cellsresults to be similar. The G79/antiTrkA treatment induced thedifferentiation of PC12 cells: 19.6±8.1 vs 11.5±2.8 for the treatmentwith only G79; for G80/antiTrkA is 14.6±4.1 15.1±6.3 for the treatmentwith only G80; for G81/antiTrkA is 16.5±5.9 vs 14.1±3.3. The percentageof differentiated cells is calculated as relative to NGF induceddifferentiation (FIG. 3H).

Example 7 Signalling Inhibition Assays G79, G80, and G81 Activate NGFDifferentiation Pathway

To evaluate if the transduction pathway induced by G79, G80, and G81involves AKT and ERK activation, PC12 cell were treated with the testcompounds in the presence of LY294002, an inhibitor of PI3K (an upstreamactivator of AKT) and PD98059, an inhibitor of MAPK kinase (an upstreamactivator of ERK). For this purpose, PC12 cells were plated intocollagen-coated 24-wells plates with 2.5% FBS and after 72 hrs the cellswere treated with the small molecules in combination with LY294002(Sigma Aldrich, 10 μM) or PD98059 (Sigma Aldrich, 50 μM). NGF (100ng/ml) was used as control and added alone or in combination with theinhibitors.

In order to assess if G79, G80 or G81 modulate TrkA signalling pathway,inhibitors of AKT and ERK pathway were used. Treatment with NGF plusLY294002 (FIG. 3D) decreased the number of differentiated cells comparedto NGF alone (6.8±2.7 vs 20.7±7.1). In the same way, the treatment withG79 plus LY294002 decreased the number of differentiated cells comparedto G79 alone (5.28±2.54 vs 11.5±2.8). Treatment with G80 plus LY294002decreased the number of differentiated cells compared to G80 alone(8.5±2.6 vs 15.1±6.3). Treatment with G81 plus LY294002 decreased thenumber of differentiated cells compared to G81 alone (6.6±4.02 vs14.1±3.3). The percentage of differentiated cells is calculated asrelative to NGF induced differentiation (FIG. 3I).

Treatment with NGF plus PD98059 (FIG. 3E) decreased the number ofdifferentiated cells compared to NGF alone (2.1±1.9 vs 20.7±7.1). In thesame way the treatment with G79 plus PD98059 decreased the number ofdifferentiated cells compared to G79 alone (3.1±1.06 vs 11.5±2.8).Treatment with G80 plus PD98059 decreased the number of differentiatedcells compared to G80 alone (4±2.3 vs 15.1±6.3). Treatment with G81 plusPD98059 decreased the number of differentiated cells compared to G81alone (1.6±1.1 vs 14.1±3.3). The percentage of differentiated cells iscalculated as relative to NGF induced differentiation (FIG. 3J).

Example 8 Binding Assay

The binding of the test compounds, G79, G80, and G81, through the TrkAor p75 receptor can be tested by a cell-based competitive ELISA. Atfirst PC12 cells are seeded into 96-well plates (Fisher Scientific) andincubated until 85%-90% of confluency. To test the binding of the testcompounds to TrkA receptor, the p75 receptor expressed on PC12 surfaceis inhibited through the binding of a blocking antibody anti-p75 (AbCam)that binds the extracellular domain of the receptor. Cells are incubatedfor 45′ with NGF at different increasing concentrations (0.01-0.1-1 nM)with or without each one of the small molecules as competitors atconstant concentration. After one wash in cool PBS the cells are fixedfor 15′ with PFA 4%. Then, cells are washed with PBS and incubated withantibody anti-NGF (1 μg/ml) for 1 h a room temperature. Cells are rinsedtwice with PBS and incubated with a secondary antibody anti-NGF for 1 hat room temperature (1:500; DyLight 488-conjugated antibody, JacksonImmunoResearch). Cells are washed and fluorescence is read by aspectrofluorimeter at 488 nm.

Example 9 Glaucoma Model G79 Prevents Neuronal Death in an Animal Modelfor Glaucoma

12 Sprague Dawley rats (4 months of age) were anesthetized withisobutane and subjected to hypertonic saline solution injection into theepiscleral vein of the right eye. Intraocular pressure (TOP) wasmeasured before the operation and was monitored one time a week using aTonoLab for 7 weeks. Treatment with G79 was begun one week afterglaucoma induction by topical application at the conjunctive. Twodifferent experiments were performed.

In the first experiment, G79 was dissolved into physiological solutionand was used at two different concentration (200 μg/ml and 400 μg/ml).NGF was used as positive control (200 μg/ml) and the physiologicalsolution, used to dissolve all the molecules, was subministered asplacebo. The animals were divided into 4 groups (3 animals in eachgroup): glaucoma-G79 200 μg/ml; glaucoma-G79 400 μg/ml; glaucoma-NGF;glaucoma-placebo. The left eye was used as the control without glaucoma.In both experiments, seven weeks after glaucoma induction, animals weresacrificed by overdose of anaesthetic and their eyes were taken andfixed in 4% of PFA. The eyes were included in paraffin and cut into 20μm sections to be used for histological studies (hematoxilin-eosinstaining). The cell count of the number of retinal ganglion cells (RGC)was performed randomly in ten different fields for each eye.

Rats were injected with a hypertonic solution in the eye that inducedhigh intraocular pressure for five weeks. Animals were treated with eyedrops of NGF (200 μM/ml), G79 (200-400 μM/ml) or placebo every day forfive weeks. Animals with glaucoma and treated with placebo (saline) havea significant decrease in the number of retinal ganglion cells comparedwith control. By contrast, animals treated with NGF drops as well asanimals treated with G79 have a significant protection of the ganglioncells (FIG. 4A). The results suggest that G79 treatment exertsneuroprotection on retinal ganglion cells (RGC).

In the second experiment, G79 at the concentration of 200 μg/ml wasused. The objective of this second experiment was to compare theefficacy of G79 to Timolol, the most common therapy for glaucoma thatworks by decreasing the intra-ocular pressure. Also in this experiment,a combinatory treatment with G79 and Timolol was performed. The animalswere divided into 4 groups having 4 animals in each group: glaucoma-G79(200 μg/ml) (n=4); glaucoma-Timolol (n=4); glaucoma-G79/Timolol (n=4);glaucoma-placebo (n=4). The left eye was used as the control withoutglaucoma. The results are shown in FIG. 4C, where G79 was compared tothe current therapy for IOP in glaucoma, i.e., Timolol. IOP inductionreduced significantly the number of RGC and this reduction wassignificant compared to the control eyes (C) (p<0.0001) and to all typeof tested treatment (p<0.0001). The combination treatment did not exertadditive effect in protecting RGCs (**GL/Timolol+G79 vs Ctr p=0.02;§GL/Timolol vs GL/Timolol+G79 p=0.003).

Example 10 Parkinson's Disease In Vitro Model (MPP Stress) G79, G80, andG81 Prevent Neuronal Death in an In Vitro Model of Parkinson's Disease

Cell culture: The human neuroblastoma cell line SH-SY5Y was maintainedin culture with 50% Ham's F12 medium and 50% EMEM medium, supplementedwith 10% FBS, 2 nM L-glutamine and 1% penicillin/streptomycin. The cellculture was maintained in a humidified atmosphere of 95% air and CO₂ at37° C.

The human neuroblastoma cell line SH-SY5Y was used to study theneuroprotective effect of the small molecules in Parkinson's disease.The SH-SY5Y cells after differentiation to neuronal phenotype withretinoic acid, were pre-treated for 3 hours with the test compounds atdifferent concentrations (20 ng/ml, 100 ng/ml, 2 μg/ml, 20 μg/ml and 50μg/ml) or BDNF (20 ng/ml) as a positive control. Then1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (100 μM) was addedand incubated for 24 hrs. The number of surviving cells was determinedthe day after by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.

The human neuroblastoma cell line SH-SY5Y was exposed to MPTP, whichdamage neurons through oxidative stress. Cells were treated with BDNF,G79, G80, G81 or placebo every day for five weeks. Cells treated withplacebo (saline) have a significant loss of cells compared with control.By contrast, cells pretreated with BDNF as well as with G79 have asignificant protection of neuronal cells (FIG. 5A).

Example 11 Oxidative Stress in the Human Cell Line SH-SY5Y

The human neuroblastoma cell line SH-SY5Y was used to study theneuroprotective effect of the test compounds G79, G80, and G81 inoxidative stress. The cells were pre-treated for 3 hours with the testcompounds at different concentrations (20 ng/ml, 100 ng/ml, 2 μg/ml, 20μg/ml and 50 μg/ml) or BDNF (20 ng/ml) as a positive control. Then H₂O₂(100 μM) was added and incubated for 24 hrs. The number of survivingcells was determined by MTT assay. A protective effect was found of G79,G80 and G81 when using H₂O₂ for inducing oxidative stress in the SH-SY5Yneuronal cell line, with a more robust antioxidative effect of G79 thanBDNF (FIG. 5B). In FIG. 5B, BDNF cell viability has been considered asthe 100%. The H₂O₂ stress induced a decrease in the percentage of cellviability (no stress Ctr vs H₂O₂ stress=100±2% vs 95.9±0.5%). G79,compared to G80 and G81, is the one that exerted better protection, atthe concentration of 20 ng/ml (BDNF vs G79=100±2.6 vs 107.4±5.8).

Example 12 Western Blot Analysis and Luminex Signalling Assay G79Activates the Neurotrophin Pathway

Western blot analysis: Neurotrophin receptors TrkA and TrkB activation(phosphorilation) was evaluated by Western-blot as follows: PC12 cellgrowing on 24-well plates were treated with low serum medium (0.5% FBS)to reduce the basal level of phosphorilation. After 24 h, the PC12 cellswere treated with G79 (100 ng/ml), then recovered and lysated in 300 μlof ice-cold lysis buffer (Sigma Aldrich) at 5-15-30-60 min. Aftersonication, cell debris were removed by centrifugation (14000 rpm for 10min) and equal amount of supernatant (30-35 μg/well) were loaded andseparated by 10% polyacrilamide gel electrophoresis (PAGE) and thenelectrobloted onto PVDF membranes. Membranes were blocked by 5% nonfatmilk and then sequentially incubated with the primary antibodies[phospho-TrkA, Tyr490 (Cell Signaling) or phospho-TrkB, Tyr515(NovusBiological)] 1:1000 over-night and the secondary antibodies1:2000, for 1 h at room temperature. Blots were processed forvisualization by chemiluminescence system (Amersham) and exposed toKodak film to visualize the fluorographic image. As shown in FIG. 6,both receptors were phosphorilated already after 5 min.

Luminex signalling assay: The human neuroblastoma cell line SH-SY5Y wasused to perform Luminex signalling assays using the 10-Plex MAPK/SAPKSignaling Kit-Phosphoprotein (Millipore). This cell line expresses theTrkB receptor and the MAPK/SAPK signalling pathway is typicallyactivated by the tyrosinkinase receptors. The phosphoprotein antibodiesincluded into the kit are the following: ERK/MAP kinase (Thr185/Tyr187);STAT-1 (Tyr701); JNK (Thr183/Tyr185); MEK-1 (Ser212); ATF-2 (Thr69/71);p53 (Ser15); HSP27 (Ser78); c-Jun (Ser73); p38 (Thr180/Tyr182), p70 S6kinase (Thr412); IkBα (Ser32) (purchased from Millipore).

The cells were propagated and then seeded into 24-well plates (30.000cell/well). After 24 hours, the cells were treated with two differentconcentrations of NGF-receptor agonist G79: 100 ng/ml and 20 μg/ml.After adding G79 into the medium, the cells were collected in the lysisbuffer of the kit at the following time points: 30 min, 1 h, 2 h, and 6h. The assay was performed in this way to assess both time and doseeffects comparing the treated samples with the untreated cells used asnegative controls (unstimulated SH-SY5Y cells). BDNF was used aspositive control at the concentration 20 ng/ml, which is the bestworking concentration known in literature. In the Luminex plate, eachsample was added into a well in duplicate. After adding all the samples,the specific positive and negative controls of phosphorilation, includedin the Luminex kit, were added. The assay buffer was also added induplicate, as a background controls.

FIG. 7 shows the intracellular pathway activated by the phosphorilationof the receptor tyrosine kinase (RTK). In the circles are the activatedfactors, while in the grey circles are the tested factors. Its effectsof the SY-SY5Y cell line in the phosphorilation of several pathways wereassessed using xMAP assay from Luminex. FIG. 8 shows the levels ofactivation of the phosphoproteins tested by Luminex technology. Thelevels of activation are expressed as Median Fluorescence Intensity (MI,I). As tested by Luminex technology, ATF-2, HSP-27, JNK and STAT-1resulted to be modulated and significantly phospho-activated compared tothe unstimulated control. FIG. 8 shows the graphics of the MFI for bothG79 and BDNF treatments, at different time-points.

ATF-2 was significantly activated at 6 h after G79 treatment (20 μg/ml),compared to unstimulated control (*p<0.05). The BDNF control showssignificant ATF-2 activation at 2 h after treatment (§p<0.05). HSP-27was significantly activated at 1 h after G79 treatment (both at 20 μg/mland 100 ng/ml), compared to unstimulated control (*p<0.05). The BDNFdoes not activate HSP-27. JNK was significantly activated at 6 h afterG79 treatment (both at 20 μg/ml and 100 ng/ml), compared to unstimulatedcontrol (* and §p<0.05). The BDNF control showed significant JNKactivation at 2 h after treatment (

p<0.05). STAT-1 was significantly activated at 6 h after G79 treatment(20 μg/ml), compared to unstimulated control (* p<0.05). The BDNF didnot activate STAT-1.

Example 13 Cytometry Binding Assay

G79 Competes with NGF and BDNF for the Binding to Neurotrophin Receptors

In order to assess the binding of G79 to the TrkA (PC12 cells) and TrkBreceptors (SH-SY5Y, we performed competitive binding assays by flowcytometry. In order to assess the binding competition between p75 andTrkA, PC12 cells expressing both TrkA and p75 receptors were cultivatedin normal growth medium. After detaching cells by trypsin solution,200,000 cells/well were added to tubes for the following incubations.One tube contains control untreated cells. In the other tubes, cellswere pre-incubated for 45 minutes with different concentrations of NGF(0-10-20-50-100 ng/ml), that can directly bind both TrkA and p75receptors, blocking the possible binding of G79 if it competes for thesame receptors. Then, without washing the cells, G79 conjugated withFITC fluorescence (100 ng/ml) was added and so incubated the cells for 1hour. Samples were read in a cytometer (FCAScan).

To detect the binding separately on both receptors, a prior incubationof cells (before NGF incubation) with blocking antibodies was made. Thecells were preincubated for 1 hour with blocking antibody anti-p75(1:100; Chemicon Int.), to detect the binding on TrkA, or blockingantibody anti-TrkA (1:200; AbCam) to detect the binding on p75. The sameexperimental protocol was repeated with SH-SY5Y cell line to detect thebinding with TrkB and p75 on these cells. In this case BDNF was used asa competitor for the binding on TrkB and p75. All the results areexpressed as mean channel fluorescence (MCF), which is a measure of thecell surface fluorescence produced by G79-FITC binding.

FIG. 9A shows that signal (MCF) decreases in presence of growingconcentration of NGF, meaning that G79 competes for the binding on thesame NGF-receptors. In order to assess the binding on p75, cells werepre-incubated with anti-TrkA antibody, wherein it was found that signal(MCF) steel decreases, which indicates that G79 could bind on p75receptor (FIG. 9B). In order to assess the binding on TrkA,pre-incubation with the antibody anti-p75 did not change the signal(MCF), meaning that G79 apparently does not compete with NGF for thebinding on TrkA (FIG. 9C). However, this result could be affected by thepresence of very small amounts of TrkA on cell surface, compared to themuch higher presence of p75 (˜75000 receptors per cell). Similarly, thebinding of G79 to TrkB was assessed using the SH-SY5Y cell line (FIG.9D). In this case, signal (MCF) decreased in the presence of growingconcentrations of BDNF, indicating that G79 competes with BDNF to one ofits receptors.

Example 14 In vitro Model of Amyotrophic Lateral Sclerosis (ALS) G79,G80, and G81 are Neuroprotective in the In Vitro Model of ALS

Cell culture: Mouse motoneuron-like cells NCS-34 were cultured in DMEM(Gibco) supplemented with 10% heat inactivated. FBS and 1%penicillin/streptomycin. The cell culture was maintained in a humidifiedatmosphere of 95% air and CO₂ at 37° C.

The nutritional deprivation stress in motorneurons was conducted asdescribed previously in Masahito T. et al., J. Neuropathol. Exp. Neurol.65(8):816-825 (2006). To assess the effect of trophic stress conditionon ALS, the presence of apoptosis was investigated using serumdeprivation. NSC-34 cells were seeded in 24-well poly-lysinated platesat 30,000 cells/well and preincubated for 24 h in DMEM plus 10% FBS withvarious doses of G79, G80, and G81 (20 ng/ml, 100 ng/ml, 2 μg/ml, 20μg/ml, 50 μg/ml), and G-CSF (2 μg/ml) or BDNF (20 ng/ml) which both wereused as positive controls (Masahito T. et al., ibid; Elliot J. L.,Neurobiology of Disease 6:310-320 (1999)). Then, the medium was removedand replaced with fresh DMEM without FBS. After 48 h, cell viability wasassayed by MTT assay, as described previously.

As shown in FIG. 10, G79, G80 and G81 exerted neuroprotection onmotorneurons challenged with serum deprivation. Cell viability isexpressed as the percentage relative to the control without stress. G79showed the best neuroprotective effect at the concentration of 100ng/ml, with a significant increase in cell viability compared tostress-Ctr (98.11±6.14% vs 69.64±10.12%; p=0.02). Also G79 showedincreased cell viability compared to both the positive controls, G-CSFand BDNF, even if not significant (G-CSF cell viability: 76.50±8.3%;BDNF cell viability: 80.62±5.2).

Example 15 Experimental Autoimmune Encephalomyelitis (EAE) Model G79Ameliorates the Animal Model of Multiple Sclerosis (Ms)

The effect of G79 was tested in the animal model of MS, the experimentalautoimmune encephalomyelitis (EAE), by performing a preventive trial (inwhich the therapy starts at the time of the induction of the disease)and a curative trial (in which the therapy starts when the animals arealready suffering from the disease). Female C57BL/6 mice from Harlan(8-12 weeks old) were immunized subcutaneously in both hind pads with300 μg of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55(Spikem, Firenze) emulsified with 50 μg of Mycobacterium tuberculosis(H37R^(a) strain; Difco, Detroit, Mich.) in incomplete Freund's adjuvant(IFA) as previously described in Palacios et al., 2007. The mice wereinjected intraperitoneally with Pertussis toxin (Sigma) (500 ng) at thetime of immunization and 2 days later. The animals were weighted andinspected for clinical signs of disease on a daily basis by a blindedobserver. The severity of the disease EAE was assessed for 30 daysaccording to the following scale: 0=normal; 0.5=mild limp tail; 1=limptail; 2=mild paraparesis of the hind limbs, unsteady gait; 3=moderateparaparesis, voluntary movements still possible; 4=paraplegia ortetraparesis; 5=moribund state. At the end of the study, the mice wereanesthetized and perfused intracardially with 4% of paraformaldehyde in0.1 M phosphate buffer (pH 7.6). The brains, spinal cords and spleenswere dissected and either fixed or frozen until use. Also serum wasobtained from all the animals included in the study. This procedure wasapproved by the University of Barcelona Committee on Animal Care.

Two different experiments were performed to assess both the preventiveeffect and curative effect of G79, compared to other drugs targetingneurotrophin pathway (Gambogic amide and Xaliproden) or one of the firstline therapy for the treatment of MS, i.e., Glatiramer acetate.

For the preventive trial, 10 animals were treated with intraperitonealinjection of G79 at the concentration of 40 mg/kg, 10 animals weretreated with intraperitoneal injection of G79 at the concentration of100 mg/kg, 10 animals were treated with intraperitoneal injection ofGambogic amide at the concentration of 2 mg/kg, 10 animals were treatedwith oral administration of Xaliproden at the concentration of 10 mg/kg.As both Gambogic amide and Xaliproden were diluted with differentpercentage of DMSO, 5 animals were treated with intraperitonealinjection of placebo (physiologic solution plus 1% DMSO) and other 5animals were treated with placebo oral (physiologic solution plus 2.5%DMSO), respectively. The treatments were performed daily, starting afterthe immunization day.

For the curative trial, to reduce the discrepancy between the oraladministration and intraperitoneal injections, as stress can deeplyaffect the development of the clinical scores in animals, all thetreatments were performed by intraperitoneal injections. For this study,8 animals were treated with G79 at the concentration of 40 mg/kg, 8animals were treated with G79 at the concentration of 100 mg/kg, 8animals were treated with Gambogic amide at the concentration of 2mg/kg, 8 animals were treated with Xaliproden at the concentration of 10mg/kg, 8 animals were treated with Glatiramer acetate at theconcentration of 5 mg/kg, 8 animals were treated with placebo(physiologic solution plus 2.5% DMSO). The treatments were performeddaily starting after the increase of the clinical score at score 2(after the second day at this score).

FIG. 11A and FIG. 11B show the results of the preventive application ofG79 in the in vivo model of MS (onset therapy in the same day of diseaseinduction). The graphics shows the clinical score of the mice affectedby EAE and treated with different drugs since the day of theimmunization. In FIG. 11A, the animals treated with G79 show a delay inthe presence of the disease, although not significant. In particular,the concentration of 100 mg/Kg was more effective in delaying thedisease. Also the final clinical score, at day 30^(th), of the animalstreated with G79 100 mg/Kg was lower compared to placebo treatedanimals.

FIG. 12 shows the results of the curative trial. G79 at the dose of 100mg/Kg, shows the best therapeutic effect in ameliorating the clinicalscore. In particular G79, compared to placebo, decreased the clinicalscore significantly, between the day 16^(th) and 23^(rd) (16^(th) day:G79 score 2.8±1.2 vs placebo score 4±0.7, p=0.01; day 19^(th): G79 score1.75±1 vs placebo score 3.3±0.5, p=0.003; G79 score 1.6±0.8 vs placeboscore 2.8±0.8, p=0.02). Also G79 100 mg/Kg exerted a better therapeuticeffect compared to the other tested treatments (Gambogic amide,Xaliproden, Glatiramer acetate).

Example 16 Neuroinflammation In Vitro Model G79 ReducesNeuroinflammation in an In Vitro Model of Neuroinflammation

Brain inflammation is a common process in many neurological diseases andit is prominent in the case of MS. In order to assess the effect of G79in brain inflammation, its effect was tested in an in vitro model ofneuroinflammation using organotypic cerebellar cultures challenged withendotoxin. First, the effect of G79 was tested in the induction of theenzyme iNOS, which produce nitric oxide and promoted inflammation. Asshown in FIG. 13, G79 decreased the expression of iNOS. Samplespre-treated with G79 showed a decrease in the expression of iNOS 24 hafter the challenge with LPS, compared to the placebo pre-treatedsamples.

Regarding the effect of G79 in the release of pro-inflammatorycytokines, the levels of TNFα and IL-1β were tested in the supernatantsfrom organotypic cerebellar cultures challenged with LPS. FIG. 14A showsthe production of TNFα in cerebellar organotypic culture. The productionof TNFα was reduced at 6 and 12 hours in organotypic culture pre-treatedwith G79 compared to placebo. FIG. 14B shows the production of IL-1β incerebellar organotypic culture. The pre-treatment with G79 did notaffect the release of IL-1β.

In vitro model of neuroinflammation in cerebellar organotypic sliceculture: Neonates mice (day 8 after birth (P8)) were decapitated afteranaesthetic IP injection and whole brains were removed aseptically.Cerebellum was separated from the rest of the brain and placed on ametal vibratome plate. Once the cerebellum was attached to the surfaceof the plate, 400 μm sagittal sections were cut using the vibratome. Theslices were then transferred with a plastic Pasteur pipette to a cellculture plate containing an organotypic culture medium (5% CO₂ in 50%basal medium with Earle's salt, 25% Hank's buffered salt solution, 25%inactivated horse serum, 5 mg/ml glucose, 0.25 mM L-glutamine and 25μg/ml penicillin/streptomycin). After separating and isolating eachslice, the cerebellar slices were then transferred to 6-well plates (3slices for each well) containing a 30 mm culture plate insert with 0.4μm pores (Millipore) and 1 ml full culture medium, pre-conditioned byincubation for at least 2 hours at 37° C., and 5% CO₂ was added intoeach well below the insert. The six-well plates were kept at 37° C. andunder 5% CO₂, and half the medium was replaced every 2 days. All theexperiments were performed after 1 week of culture in such conditions.

After one week of organotypic cerebellum culture, the medium of eachwell was replaced with fresh medium and G79 (100 ng/l) or placebo(physiologic solution) was added to the wells and kept in incubation for1 hour. Then lipopolysaccharide (LPS) (15 μg/ml) was added and kept inincubation. Slices and organotypic culture medium were recovered atdifferent time points: Oh, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h. For eachtime point, untreated slices of control, LPS/placebo treated slices anda LPS/G79 treated slices were obtained. Slices were collected in threedifferent experiments for RNA extraction. For this purpose, slices wererecovered directly in RNA Lysis Buffer (Qiagen) and frozen at −20° inthe same buffer. In the other three different experiments, slices wererecovered for immunofluorescence and fixed in 4% paraformaldehyde (PFA)for 45 minutes at room temperature. In all the experiments, organotypicculture medium was collected and stored at −20° for ELISA assay.

Real-time quantitative polymerase chain reaction: Organotypic slicescollected from the LPS stimulation experiment were homogenized in RNAlysis buffer. Total RNA was extracted using the RNeasy Mini Kit (Qiagen,Chatwworth, Calif.) isolation system, including DNase treatment usingthe RNase-Free DNase Set (Quiagen). Total RNA (35 pig) was reversetranscribed using the Reverse Transcription System (High Capacity cDNAArchive Kit; Applied Biosystems, Foster City, Calif.). The real timereaction was conducted at 25° C. for 10 minutes, followed by 37° C. for2 hours, and finally stored at 4° C. Primers and target-specificfluorescence-labeled TaqMan probes were purchased from AppliedBiosystems (TaqMan Gene Expression assays). The primer for the gene ofiNOS was used. The TaqMan Universal Master Mix (Applied biosystems) wasused. Amplification of complementary DNA was performed on a DNA EngineOpticon 2 Real-Time System (MJ Research, Watertown, Mass.) using 0.9 μMfor each primer and 0.25 μM for the probe and 20 ng complementary DNA.The reaction conditions were as follows: an initial 2 minutes at 50° C.,followed by 10 minutes at 95° C., and 40 cycles of 15 seconds at 95° C.,and 1 minute at 60° C. Each sample was run in triplicate, and in eachplate the target and the endogenous control were amplified in the samewell. The expression of the gene tested was quantified relative to thelevel of the housekeeping gene GAPDH.

ELISA assay: The supernantants from cerebellar organotypic culturemedium were used to assay cytokine production such as IL-1-β and TNF-αby ELISA. For IL-1-β, Quantikine Immunoassay IL-1-13 kit (R&D System)was used, and for TNF-α the ELISA Development kit for mouse TNF-α(Peprotec) was used. All the ELISA assays were performed according themanufacture's instructions.

Example 17 Transport Through the Blood-Brain Barrier (BBB)

The ability of the compounds G79, G80 and G81 to cross the blood-brainbarrier was tested using two in vitro models. The passive transport wastested using the PAMPA (Parallel Artificial Membrane Permeability Assay)model and the active transport was tested using an in vitro model of theBBB by co-culturing BBEC and astrocytes. As a result, it was found thatG79 was unable to cross the BBB in the PAMPA model compared to otherdrugs with a good ability for crossing in the PAMPA model, such aspropanolol and carbamazepine (Effective permeability (pe)propanolol=11.5; pe carbamazepine=10.3; pe G79=0×10⁻⁶ cm/s). Bycontrast, in the in vitro cell model of the BBB, all three moleculesdisplayed medium to high crossing of the BBB by active transport (peG79=4.1; pe G80=2.8; pe G81=2.1×10⁻⁶ cm/s).

Parallel Artificial Membrane Permeability Assay (PAMPA) assay: PAMPA isused as an in vitro model of passive BBB permeability. An artificialmembrane immobilized on a filter is placed between a donor and acceptorcompartment. At the start of the test, a drug is introduced in the donorcompartment. Following the permeation period, the concentration of thedrug in the donor and acceptor compartments are measured using UVspectroscopy. Therefore the permeability of any compound with a UVchromophore can be determined by this method.

The tested compound stock solutions were diluted 200-fold in universalbuffer at pH 7.4 and added to the donor wells. The filter membrane wascoated with PBL in dodecane and the acceptor well was filled with pH 7.4buffer. The acceptor filter plate was carefully put on the donor plateto form a ‘sandwich’ (consisting of the aqueous donor with test thecompound on the bottom, an artificial lipid membrane in the middle, andan aqueous acceptor on the top). The test Compound diffused from thedonor well through the lipid membrane and into the acceptor well. The‘sandwich’ was left undisturbed for 18 hrs while the permeationoccurred. The concentration of the test compound in the acceptor, thedonor, and the reference wells was determined using the UV plate reader.Effective permeability (Pe) of each compound was calculated by using thepION PSR4p software. Samples were analyzed in triplicate and the averageof the three runs was reported. Quality control standards were run witheach sample set to monitor the consistency of the analysis set.

Cellular in vitro model of transport through the blood-brain barrier(BBB): The cellular in vitro model was established by using a co-cultureof blood-brain endothelial cells (BBECs) and newborn rat astrocytes. Inbrief, before cell co-culture (24-well polycarbonate transwell with asurface area of 0.33 cm² and pore-size of 0.4 μm, Corning Costar), theupper surface of plate inserts was coated with collagen type IV andfibronectin. Next, the inserts were placed upside down in a large petridish and 40 μL of a suspension (containing approximately 45,000astrocytes) was placed on the bottom of each filter. The Petri dish wasplaced in an incubator for 1 hour and 40 μL of fresh DMEM+S was added tothe bottom of each filter every 15 minutes. The inserts were thentransferred back into the plate and incubated at 37° C., 5% CO₂ forthree days. After this time, 2 hours before seeding the BBECs, themedium was replaced by DMEM+S supplemented with 125 μg/ml of heparin.Two hours later, cells were seeded in the inserts (45,000 cells perfilter). The plate was kept in the incubator at 37° C., 5% CO₂ for threemore days. After three days of co-culture, the medium was replaced byDMEM+S supplemented with cAMP and RO-20-1724, and kept at 37° C. and 5%CO₂. On day 8 of co-culture, transendothelial electrical resistance(TEER) measures showed that the system was ready for transport studies.To validate the maturity of the model, on the same day of theexperiment, permeability assays were done in parallel with luciferyellow (LY) as an integrity marker of the in vitro barrier, During thepermeability assay the samples were co incubated with LY at aconcentration of 20 μM to assess the integrity of the cellular monolayerduring the assay.

The TEER was determined by using an ohmmeter Millicell ERS system (MFRS000 01, Millipore), TEER measures confirm formation of a functionallyintact in vitro BBB by day 8 of co-culture. The TEER values representthe tightness or integrity of the in vitro BBB. The TEER value (mean±SD)for all wells was 141±5.7 ohms/cm².

Papp=(dQ/dt)*(1/A)*(1/C ₀)(cm/s)  (1)

In the above equation (1), (dQ/dt) is the amount of the compound presentin the acceptor compartment in function of time (nmol/s), A is the areaof the insert (cm²) and C_(o) is the initial concentration of thecompound applied to the donor compartment (nmol/ml).

During transport studies, compounds were co-incubated with LY (20 μM) inorder to ensure the integrity of the cell membrane during the transportassay.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All patents and publications cited herein are fully incorporated byreference herein in their entirety.

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1. (Original A compound having Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R₁ isphenyl substituted with halogen or trifluoromethyl, and furtheroptionally substituted with one or two substituents selected from thegroup consisting of halogen, C₁₋₆ alkyl, (C₁₋₆)alkoxy, andhalo(C₁₋₆)alkyl; or R₁ is pyrrolidin-1-yl; R₂ is2-oxo-pyrrolidin-1-ylmethyl or sulfamoylphenyl; and R₃ is chosen frompropyl, 1-methylethyl, butyl, 2-methylpropyl, pentyl, 1-methylbutyl,2-methylbutyl, hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl,and 1-methylpentyl.
 2. The compound of claim 1, wherein R³ is2-methylpropyl, having the Formula II:

or a pharmaceutically acceptable salt or prodrug thereof, wherein R₁ andR₂ are as defined in claim
 1. 3. The compound of claim 1, wherein R₁ isfluorophenyl and R₂ is as defined in claim
 1. 4. The compound of claim3, wherein R₁ is 2-fluorophenyl.
 5. The compound of claim 1, wherein R₁is pyrrolidin-1-yl.
 6. The compound of claim 1, wherein R₂ is2-oxo-pyrrolidin-1-ylmethyl.
 7. The compound of claim 1, wherein R₂ issulfamoylphenyl.
 8. The compound of claim 7, wherein R₂ is4-sulfamoylphenyl.
 9. The compound of claim 2, having the Formula III:

or a pharmaceutically acceptable salt or prodrug thereof.
 10. Thecompound of claim 2, having the Formula IV:

or a pharmaceutically acceptable salt or prodrug thereof.
 11. Thecompound of claim 2, having the Formula V:

or a pharmaceutically acceptable salt or prodrug thereof. 12-25.(canceled)
 26. A pharmaceutical composition, comprising atherapeutically effective amount of at least one compound of claim 1,and a pharmaceutically acceptable carrier. 27-39. (canceled)
 40. Amethod of prevention or treatment of nerve cell death or damage,comprising administering to a subject in need thereof an effectiveamount of the compound of claim 1, or a pharmaceutically acceptable saltor prodrug thereof.
 41. A method of neuroprotection, comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 1 or a pharmaceutically acceptable salt or prodrugthereof.
 42. A method of neuroprotection and immunomodulation,comprising administering to a subject in need thereof an effectiveamount of the compound of claim 1, or a pharmaceutically acceptable saltor prodrug thereof.
 43. A method of regenerating nerve cells, comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 1, or a pharmaceutically acceptable salt or prodrugthereof.
 44. A method of prevention or treatment of a disease,comprising administering to a subject in need thereof an effectiveamount of the compound of claim 1, or a pharmaceutically acceptable saltor prodrug thereof, wherein the disease is selected from: neurologicaldiseases, preferentially neurodegenerative disorders, such asamyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, Friedreich's ataxia, Huntington's disease, Dementia with Lewybodies, and spinal muscular atrophy; nerve inflammation, such asmultiple sclerosis or neuromyelitis optica; major depressive disorder;schizophrenia; glaucoma; peripheral neuropathy, such as diabetic or AIDSneuropathy; and cancer, such as glioblastoma, astrocytoma,meduloblastoma, neurinoma, neuroblastoma, meningioma, colon cancer,pancreatic cancer, breast cancer, prostate cancer, leukemia, acutelymphocytic leukemia, osteosarcoma, hepatocellular carcinoma, ovariancarcinoma, lung adenocarcinoma, and esophagic carcinoma.
 45. The methodof claim 44, wherein the disease is multiple sclerosis.
 46. The methodof claim 44, wherein the disease is Alzheimer's disease.
 47. The methodof claim 44, wherein the disease is Parkinson's disease.
 48. The methodof claim 44, wherein the disease is glaucoma.
 49. A method of treating adisease responsive to the stimulation of the activity of nerve growthfactor, or a nerve growth factor receptor, in a mammal suffering fromlack of stimulation thereof, comprising administering an effectiveamount a compound claim 1, or a pharmaceutically acceptable salt orprodrug thereof.
 50. (canceled)
 51. A method of stimulating nerve growthfactor receptor activity in a subject in need thereof, comprisingadministering a compound of claim 1, or a pharmaceutically acceptablesalt or prodrug thereof.
 52. The method of claim 51, wherein the nervegrowth factor receptor is TrkA receptor or p75 receptor.
 53. A method oftreating a disease responsive to the stimulation of the activity ofneurotrophin factor, or a neurotrophin factor receptor, in a mammalsuffering from lack of stimulation thereof, comprising administering aneffective amount a compound of claim 1, or a pharmaceutically acceptablesalt or prodrug thereof.
 54. (canceled)
 55. A method of stimulatingneurotrophin factor receptor activity in a subject in need thereof,comprising administering a compound of claim 1, or a pharmaceuticallyacceptable salt or prodrug thereof.
 56. A method of treating a diseaseresponsive to the stimulation of the activity of brain-derivedneurotrophic factor, or a brain-derived neurotrophic factor receptor, ina mammal suffering from lack of stimulation thereof, comprisingadministering an effective amount a compound of claim 1, or apharmaceutically acceptable salt or prodrug thereof.
 57. (canceled) 58.A method of stimulating brain-derived neurotrophic factor receptoractivity in a subject in need thereof, comprising administering acompound of claim 1, or a pharmaceutically acceptable salt or prodrugthereof.
 59. The method of claim 58, wherein the brain-derivedneurotrophic factor receptor is TrkB receptor or p75 receptor.
 60. Amethod of preparing a pharmaceutical composition, comprising admixing aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt or prodrug thereof, with a pharmaceutically acceptablecarrier.
 61. A method of prevention or treatment of nerve cell death ordamage, comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition of claim
 26. 62. Amethod of neuroprotection, comprising administering to a subject in needthereof an effective amount of the pharmaceutical composition of claim26.
 63. A method of neuroprotection and immunomodulation, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition of claim
 26. 64. A method of regeneratingnerve cells, comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition of claim
 26. 65. Amethod of prevention or treatment of a disease, comprising administeringto a subject in need thereof an effective amount of the pharmaceuticalcomposition of claim 26, wherein the disease is selected from:neurological diseases, preferentially neurodegenerative disorders, suchas amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer'sdisease, Friedreich's ataxia, Huntington's disease, Dementia with Lewybodies, and spinal muscular atrophy; nerve inflammation, such asmultiple sclerosis or neuromyelitis optica; major depressive disorder;schizophrenia; glaucoma; peripheral neuropathy, such as diabetic or AIDSneuropathy; and cancer, such as glioblastoma, astrocytoma,meduloblastoma, neurinoma, neuroblastoma, meningioma, colon cancer,pancreatic cancer, breast cancer, prostate cancer, leukemia, acutelymphocytic leukemia, osteosarcoma, hepatocellular carcinoma, ovariancarcinoma, lung adenocarcinoma, and esophagic carcinoma.